Antibody against fibroblast growth factor-23

ABSTRACT

The purpose of the present invention is to provide an antibody against fibroblast growth factor 23. The antibody is obtained by immunizing an animal with a polypeptide which comprises an amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 1 by deletion, substitution, or addition of 1 or several amino acids, and has fibroblast growth factor-23 activity and activity to control phosphate metabolism or vitamin D metabolism, and is shown by the following (a), (b), or (c): 
         (a) an antibody, which recognizes an amino acid sequence between the 180 th  and the 194 th , or the 237 th  and the 251 st  amino acid residues represented by SEQ ID NO: 1; (b) an antibody, which is produced by a hybridoma whose accession number is FERM BP-7838, FERM BP-7839, FERM BP-7840, or FERM BP-8268; or (c) an antibody, which is competitive with the antibody produced by the hybridoma whose accession number is FERM BP-7838, FERM BP-7839, FERM BP-7840, or FERM BP-8268 upon binding with the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1.

TECHNICAL FIELD

The present invention relates to antibodies against a fibroblast growthfactor-23 (FGF-23).

More particularly, the present invention relates to antibodies thatprovides a method for diagnosing diseases or pathological conditionsaccompanied by accumulation of or decreases in an FGF-23 protein in vivoby properly detecting and measuring FGF-23, and that enables improvementor treatment of the conditions of diseases or pathological conditionsresulting from the excessive action of FGF-23 by suppressing FGF-23, andalso relates to a method for preparing and a method for using anantibody.

BACKGROUND ART

Fibroblast growth factor was purified for the first time from thehypophysis of cattle as a substance that stimulates the growth of afibroblast line NIH3T3. Thereafter, analogous proteins were identifiedin various tissues, and a group of these substances forms a polypeptidefamily (FGF family). To date, 22 types of protein belonging to the FGFfamily have been identified in vertebrates. As the biological activitiesof these types of protein, not only the fibroblast-growth stimulatingactivity, but also a wide range of actions are known, such as growth ofthe mesoderm and the neuroectoderm, angiogenesis, and limb bud formationin the developmental stage. FGF family members vary in terms ofexpression sites and expression times of genes. The genes thereof areoften expressed only at specific sites in the developmental stage or inadults. As genes encoding the receptors of FGF, at least 4 types areknown: FGFR1, FGFR2, FGFR3, and FGFR4. In addition, it is known that inFGFR1, FGFR2, and FGFR3, receptor proteins differing in terms of theirextracellular domains are independently present due to differences insplicing. Furthermore, it is known that heparin and heparan sulfateproteoglycan interact with FGF and FGF receptors, so as to regulate theaction. Furthermore, there are many proteins belonging to the FGF familybecause of structural similarity, but their biological activities, theirreceptor-binding abilities, and the like remain almost unknown.Characteristics of the FGF family have been completed as reviews (OrnitzD. M. and Itoh N. Fibroblast growth factors. Genome biology 2: 3005.1-3005. 12, 2001).

FGF-23 was cloned for the first time from a mouse by data base searchutilizing its homology with FGF-15 and the PCR method, and then humanFGF-23 was cloned utilizing its sequence homology with that of mouseFGF-23 (Yamashita T., Yoshioka M., and Itoh N. Identification of a NovelFibroblast Growth Factor, FGF-23, Preferentially Expressed in theVentrolateral Thalamic Nucleus of the Brain. Biochem. Biophy. Res.Commun. 277: 494-498, 2000). Subsequently, in research on autosomaldominant hypophosphatemic rickets/osteomalacia (hereinafter referred toas ADHR), missense mutations were characteristically discovered in theFGF-23 genes of ADHR patients while narrowing the region of mutant genesin ADHR patients and identifying responsible genes (The ADHR Consortium.Autosomal dominant hypophosphatemic rickets is associated with mutationsin FGF-23. Nature Genet. 26: 345-348, 2000). This discovery has stronglysuggested physiological importance of FGF-23 in vivo. However, thebiological activities of FGF-23 have remained unknown. In the meantime,the biological activity of FGF-23 has been determined by research ontumor-induced osteomalacia. It has been thought that in this disease, atumor responsible for the disease produces and secretes a humoral factorinducing the disease, and by the action of this factor, morbidity suchas hypophosphatemia or osteomalacia is developed.

In search of this disease-inducing factor produced by such responsibletumor, FGF-23 has been cloned as a gene that is expressed at high levelsin tumors. Furthermore, it has been shown that by the administration ofthis factor, hypophosphatemia and osteomalacia are reproduced (ShimadaT., Mizutani S., Muto T., Yoneya T., Hino R., Takeda S, Takeuchi Y.,Fujita T., Fukumoto S and Yamashita T., Cloning and characterization ofFGF23 as a causative factor of tumor-induced osteomalacia. Proc Natl.Acad. Sci. 98: 6500-6505, 2001). This research has shown the involvementof FGF-23 in in vivo metabolic control relating to phosphorus andcalcium, and suggested that FGF-23 acts as a systemic factor expressingits action while circulating in vivo. However, the in vivo concentrationand metabolism required for the expression of the action of FGF-23 havenot been shown, and the physiological role of FGF-23 remains largelyunknown. Moreover, as a disease presenting conditions analogous inclinical findings, X-linked hypophosphatemic rickets is known. However,the involvement of FGF-23 in the morbidity has not been revealed. Exceptfor ADHR and tumor-induced osteomalacia, there are no known diseasesthat have been proven to be associated with FGF-23.

The above tumor-induced osteomalacia is characterized by showingabnormally low levels of blood phosphorus and 1α,25-dihydroxyvitamin D(hereinafter referred to as 1,25D) along with tumorigenesis. It isaccompanied by decreased muscle force, or osteomalacia, and may resultin dysbasia or dysstasia. In most cases, the responsible tumor for thisdisease is a benign tumor derived from mesenchymal cells. Mostresponsible tumors are poor in growth ability, and notable increases arebarely observed during follow-up. Furthermore, although the progressionof morbidity is observed, detailed examination such as whole bodyscanning by MRI inspection is often required to find a responsibletumor. Accordingly, some cases where confirmed diagnosis is not givenand a diagnosis of hypophosphatemia with unknown causes is made aresuspected of being tumor-induced osteomalacia. Currently the only methodthat results in a confirmed diagnosis of tumor-induced osteomalacia isto confirm recovery from conditions of the disease by tumorectomy. Thisis because there are no methods for examining the cause and effectrelationship between tumorigenesis and conditions of disease by clinicaltests. In some cases, removal of non-responsible tumors that arecompletely independent from conditions of disease has been conducted. Toimprove such circumstances, development of a method for clinical testswhereby differential diagnosis can be made for tumor-inducedosteomalacia has been expected.

The fact that FGF-23 has action controlling in vivo phosphorusmetabolism has been discovered. Parathyroid hormones and 1,25D that havebeen known to have action controlling phosphorus metabolism play a moreimportant role in controlling calcium metabolism rather than inphosphorus metabolism. No molecules mainly controlling phosphorusmetabolism have been known, and FGF-23 is expected to have suchactivity. In the meantime, a close relationship between phosphorusmetabolism and calcium metabolism is clinically known. In particular, interms of calcification of bone tissues and pathological ectopiccalcification, it is difficult to consider the two separately. Based onthe facts that FGF-23 in an excessive state induces osteomalacia andFGF-23 has action lowering 1,25D, FGF-23 may be involved not only inphosphorus metabolism but also extensively in controlling calcificationand bone metabolism. Moreover, some of diseases with abnormalities inorgans controlling mineral metabolism consisting mainly of theintestinal tract, the kidney, and the bone tissues may be associatedwith the excessive accumulation and the deficiency state of FGF-23.Development of a method for testing in vivo concentrations of FGF-23 isalso expected to result in an understanding of such diseases, and inprecise treatment of the diseases.

Suppression or removal of FGF-23 in morbidity induced by excessiveFGF-23 can be a therapeutic method for the disease. One possible methodis the inhibition of ligand-receptor interaction using an antagonist forthe receptor of FGF-23 or a substance binding to FGF-23, and anotherpossible method involves the removal of FGF-23 using a substance bindingto FGF-23. However, there are no known substances that selectivelysuppress or remove FGF-23 by the above-mentioned methods.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an antibody recognizingFGF-23, and a diagnosis method, a therapeutic method, and a prophylacticmethod using the antibody against diseases in which FGF-23 is involved.

As a result of intensive studies to achieve the above objectives, wehave completed the present invention by obtaining an antibody thatspecifically recognizes and binds to a partial structure of an FGF-23protein, and discovering that FGF-23 can be detected using the antibody.

The present invention is as follows.

(1) An antibody obtained by immunizing an animal with a polypeptidewhich consists of an amino acid sequence represented by SEQ ID NO: 1, oran amino acid sequence derived from the amino acid sequence representedby SEQ ID NO: 1 by deletion, substitution, or addition of 1 or severalamino acids, and has fibroblast growth factor-23 activity, which hasactivity controlling phosphate metabolism or vitamin D metabolism, andis shown by the following (a), (b), or (c):

-   -   (a) an antibody which recognizes an amino acid sequence between        the 180th and the 194^(th), or the 237^(th) and the 251^(st)        amino acid residues represented by SEQ ID NO: 1;    -   (b) an antibody which is produced by a hybridoma whose accession        number is FERM BP-7838, FERM BP-7839, FERM BP-7840, or FERM        BP-8268; or    -   (c) an antibody which is competitive with the antibody produced        by the hybridoma whose accession number is FERM BP-7838, FERM        BP-7839, FERM BP-7840, or FERM BP-8268 upon binding with the        polypeptide consisting of the amino acid sequence represented by        SEQ ID NO: 1.

(2) A pharmaceutical composition, which comprises the above antibody asan active ingredient.

The pharmaceutical composition of the present invention may comprise anantibody that recognizes the amino acid sequence between the 180th andthe 194th amino acid residues represented by SEQ ID NO: 1. Thecomposition of the present invention is effective against at least onedisease selected from tumor-induced osteomalacia, ADHR, XLH, renalosteodystrophy, dialysis osteopathy, osteoporosis, hypophosphatemia,rickets, osteomalacia, dysfunction of the renal tubule, osteopenia,hypocalcemia, disorder of bone extension, disorder of bonecalcification, hyperparathyroidism, ectopic calcification, itching,osteosclerosis, Paget's disease, hypercalcemia, hypoparathyroidism,ostealgia, decreased muscle force, skeletal deformation, failure tothrive, and hypo-1,25D-hemia (disease characterized by low levels of1.25D in blood), and can be used for treating or preventing thesediseases.

The pharmaceutical composition comprises an agent promoting osteogenesiscomprising the above antibody as an active ingredient.

(3) A method for detecting a fibroblast growth factor-23, whichcomprises causing an antibody that recognizes a part of an amino acidsequence between the 25^(th) and the 179^(th) amino acid residuesrepresented by SEQ ID NO: 1 and an antibody that recognizes a part of anamino acid sequence between the 180th and the 251^(st) amino acidresidues represented by SEQ ID NO: 1 to react with a test sample.

An example of such an antibody (the antibody that recognizes a part ofan amino acid sequence between the 180^(th) and the 251^(st) amino acidresidues represented by SEQ ID NO: 1) used in the above detection methodis an antibody that recognizes an amino acid sequence between the180^(th) and the 196^(th) amino acid residues represented by SEQ IDNO: 1. In addition, a thrombin inhibitor can also be used in thedetection method of the present invention.

(4) A kit for detecting a fibroblast growth factor-23, which contains anantibody that recognizes a part of the amino acid sequence between the25^(th) and the 179^(th) amino acid residues represented by SEQ ID NO: 1and an antibody that recognizes a part of the amino acid sequencebetween the 180th and the 251st amino acid residues represented by SEQID NO: 1.

The kit of the present invention contains an antibody that recognizesthe amino acid sequence between the 180th and the 196th amino acidresidues represented by SEQ ID NO: 1 as an antibody that recognizes apart of the amino acid sequence between the 180th and the 251st aminoacid residues represented by SEQ ID NO: 1.

(5) An anti-fibroblast growth factor-23 antibody-binding material, towhich at least one antibody selected from the above antibodies is bound.

(6) A medical appliance, which is provided with the above bindingmaterial. The medical appliance of the present invention is used forremoving the fibroblast growth factor-23 in blood.

(7) A pharmaceutical composition, which comprises as active ingredientsat least 2 types of the above antibodies that recognize different sites.

The present invention is explained in detail as follows.

FGF-23 is known to be expressed at high levels in tumors responsible fortumor-induced osteomalacia. When cells secreting FGF-23 areexperimentally transplanted into a nude mouse, hypophosphatemia, orosteomalacia, which is a characteristic of tumor-induced osteomalacia,is reproduced. Thus, FGF-23 is thought to be an inducer of tumor-inducedosteomalacia. Independently, research and studies have been conducted ona gene responsible for autosomal dominant hypophosphatemic rickets(ADHR), which is one mode of hereditary hypophosphatemia. Thus, FGF-23has been identified as a gene whose mutations are specifically observedin ADHR patients. These results suggest the involvement of FGF-23 as apathogenic factor of hypophosphatemic diseases. For the biologicalactivity of FGF-23, it has been confirmed that FGF-23 induceshypophosphatemia in experiments where a recombinant FGF-23 isadministered to mice. Based on these results, it is assumed that FGF-23acts in vivo as a humoral factor to control phosphorus metabolism andbone metabolism. Hence, quantitative and qualitative evaluation of invivo FGF-23 is very useful in understanding and diagnosing diseases.Furthermore, controlling the biological activity of FGF-23 is expectednot only to be able to cure hypophosphatemic diseases for which theinvolvement of FGF-23 has been shown as described above, but also toenable the control of phosphorus metabolism and bone metabolism, and toapply to therapies for abnormalities in mineral metabolism, metabolicbone diseases, and the like.

The present invention will be described in detail by showing themeanings of the terms used in the present invention.

Single alphabet characters used to denote amino acids in thisspecification and in drawings of the present application are as follows:(G) glycine, (A) alanine, (V) valine, (L) leucine, (I) isoleucine, (S)serine, (T) threonine, (D) aspartic acid, (E) glutamic acid (N)asparagine, (Q) glutamine, (K) lysine, (R) arginine, (C) cysteine, (M)methionine, (F) phenylalanine, (Y) tyrosine, (W) tryptophan, (H)histidine, and (P) proline. In addition, the meaning of single alphabetcharacters used to denote the components of DNA is as follows: (A)adenine, (C) cytosine, (G) guanine, and (T) thymine.

Activity controlling phosphate refers to activity controlling phosphateconcentrations in blood.

Activity controlling vitamin D metabolism refers to changes in absolutelevels of vitamin D existing in vivo and metabolites synthesized in vivoby the use of vitamin D, or potency controlling changes in the existencerate thereof.

1. Antibody Recognizing FGF-23

Human FGF-23 used in the present invention is a polypeptide having theamino acid sequence (SEQ ID NO: 1) described below, and has theabove-illustrated characteristics (activity). Furthermore, FGF-23 and apart thereof in the present invention encompass a human FGF-23derivative having an amino acid sequence substantially the same as thatof the primary structure of a natural-type FGF-23, and a part of thehuman FGF-23 derivative, as long as the later described antibody of theinvention of the present application has reactivity.

Here, the term “human FGF-23 derivative having substantially the sameamino acid sequence” refers to a protein having an amino acid sequencederived from the amino acid sequence by substitution, deletion, and/ormodification of 1 or several amino acids, as long as it has properties(activity) substantially equivalent to those of natural-type humanFGF-23. Moreover, a plural number of such substitution, deletion,modification, and addition may be combined. The activity of the humanFGF-23 refers to activity that can induce hypophosphatemia orosteomalacia similarly to the above case.

Human FGF-23 of the present invention can be produced appropriatelyusing a method known in the technical field, such as a chemicalsynthesis method, a cell culture method, or a modified method thereof,in addition to gene recombination techniques. Moreover, a partialsequence of human FGF-23 can also be produced by gene recombinationtechniques or the chemical synthesis method according to a method knownin the technical field described below, or a modified method thereof, orcan be produced by appropriately cleaving human FGF-23 isolated by thecell culture method using a proteolytic enzyme or the like.

The antibody in the present invention is an antibody having reactivityto human FGF-23 as defined above or a part thereof, or is a part of suchantibody. The antibody of the present invention encompasses a monoclonalantibody comprising a heavy chain and/or light chain having an aminoacid sequence derived from the amino acid sequence of each heavy chainand/or light chain composing the antibody by deletion, substitution, oraddition of 1 or several amino acids, and can bind to FGF-23. Theabove-described partial alteration of amino acids (deletion,substitution, insertion, and addition) can be introduced into the aminoacid sequence of the human FGF-23 or the antibody of the presentinvention by partially altering a nucleotide sequence encoding the aminoacid sequence. Techniques for these alterations are known by personsskilled in the art, and a commercially available kit for introducingmutations or the like can be used.

(1) Antibody Specifically Recognizing and Binding to a Partial Structureof FGF-23 Protein

To obtain an antibody that is useful in detecting FGF-23 and controllingthe biological activity of FGF-23, it is effective to obtain an antibodyrecognizing the structural characteristic of FGF-23, an antibody havinghigh affinity therefor, an antibody capable of neutralizing biologicalactivity, or the like. Since the structure and antigenicity of FGF-23have been unknown, a plural number of peptides corresponding to partialsequences of FGF-23 were synthesized and antibodies against each peptidewere obtained. FGF-23 secreted by expression cells was detected byWestern blotting using these antibodies, revealing that the culturesupernatant of the expression cells contained a large quantities oflow-molecular-weight peptides derived from FGF-23 protein in addition tomature FGF-23 protein. Furthermore, the obtained antibodies exhibited avariety of binding specificities against mature- FGF-23 and peptidesgenerated by the cleavage of mature FGF-23.

Furthermore, the binding activities of the antibodies in the liquidphase were revealed by an immunoprecipitation method. Furthermore, acombined use of these site-specific antibodies made it possible todetect peptides having specific sequence regions from various peptidesderived from FGF-23. Details concerning the modification and themetabolism of FGF-23 have not been shown. We revealed, when FGF-23 isexpressed in CHO cells, the presence of not only full-length proteinlacking signal sequences, but also metabolites resulting from cleavagebetween the 179^(th) Arg residue and the 180^(th) Ser residue, bycarrying out detection experiments using various antibodies obtained inthe present invention. In addition, we have revealed that whereas inplasma FGF-23 is present in its full-length form, in serum, cleavageoccurs between the 196^(th) Arg residue and the 197^(th) Ala residue ofFGF-23, by conducting an experiment wherein blood samples of plasma andthose of serum containing FGF-23 were collected, and the metaboliteswere detected, as well as an experiment wherein the purifiedrecombinants were admixed with serum. This may be due to thrombin, andcleavage may also occur between the 198^(th) Arg and the 199^(th) Met.

(2) Production of Antibody

The antibody of the present invention can be produced by the followingproduction method, as an example. Specifically, for example, a non-humanmammal such as a human-antibody-producing transgenic mouse is immunizedwith a product bound with the above-defined human FGF-23 or a partthereof, or their binding complex with an appropriate substance (e.g.,bovine serum albumin) for enhancing the antigenicity of an antigen,together with an adjuvant (e.g., Freund's Adjuvant), if necessary.Alternatively, immunization can be conducted by administering anexpression vector having FGF-23 incorporated therein. Polyclonalantibodies can be obtained from serum obtained from immunized animals.In addition, monoclonal antibodies can be produced by preparinghybridomas from antibody-producing cells obtained from immunized animalsand myeloma cells incapable of producing antibodies by theirselves,cloning the hybridomas, and selecting clones that produce monoclonalantibodies showing specific affinity for the antigens used forimmunization.

More specifically, monoclonal antibodies can be produced as describedbelow. Hybridomas secreting monoclonal antibodies can be prepared by,and according to Köhler and Milstein et al.'s method (Nature, 1975 Vol.256: 495-497). Specifically, a hybridoma is prepared by fusingantibody-producing cells contained in the spleen, the lymph node, thebone marrow, the tonsils, or the like obtained from an animal immunizedas described above, and preferably the lymph node or the spleen, withmyeloma cells incapable of producing antibodies derived preferably frommammals such as mice, rats, guinea pigs, hamsters, rabbits, or humans.Screening for hybridoma clones producing monoclonal antibodies can beperformed by culturing hybridomas in, for example, a microtiter plate,measuring the reactivity to the immunogens in the culture supernatant inwells where growth is observed by, for example, an enzyme immunoassaymethod such as ELISA.

Monoclonal antibodies can be produced from hybridomas by culturinghybridomas in vitro and isolating antibodies from the culturesupernatant, or by culturing hybridomas in vivo, such as in the ascitesof mice, rats, guinea pigs, hamsters, or rabbits, and then isolatingantibodies from the ascites.

Moreover, recombinant antibodies can be prepared by cloning a geneencoding a human monoclonal antibody from an antibody-producing cellsuch as a hybridoma, incorporating the gene into an appropriate vector,introducing the vector into a host (e.g., mammalian cell lines,Escherichia coli, yeast cells, insect cells, and plant cells), and thencausing the production of antibodies using gene recombination techniques(P. J. Delves, ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY; PShepherd and C Dean, Monoclonal Antibodies., 2000 OXFORD UNIVERSITYPRESS, J. W. Goding, Monoclonal Antibodies: principles and practice,1993 ACADEMIC PRESS). Furthermore, transgenic cattle, goats, sheep, orpigs wherein a gene of a target antibody has been incorporated into anendogenous gene using transgenic animal production techniques areproduced, and then monoclonal antibodies derived from the antibody genecan be obtained in large quantities from the milk of the transgenicanimals. When a hybridoma is cultured in vitro, in accordance withvarious conditions including the properties of the cell type to becultured, the purpose of tests and research, culture method, and thelike, hybridomas can be grown, maintained, and stored using known typesof nutrient media that are used for producing monoclonal antibodies inculture supernatant, or any type of nutrient media induced and preparedfrom known basic media.

Regarding the polyclonal antibody of the present invention, as shown inExample 5, a chemically synthesized partial peptide of FGF-23 was boundto a bovine thyroglobulin (the carrier protein) and a rabbit wasimmunized with the product. Then antibodies, which had been induced toact against each peptide by immunization, were purified with an affinitycolumn to which the peptides used for immunization had been immobilized.The properties of the thus obtained antibodies were examined by Westernblotting and ELISA, so that the reactivities against the FGF-23 proteinwere made clear.

Regarding the preparation of the monoclonal antibody of the presentinvention, immunization was performed by two methods shown in Example 3.The properties of monoclonal antibodies produced by the thus obtainedhybridomas, that is, reactivities against immobilized FGF-23 partialpeptides shown in Example 9 and reactivity against the FGF-23 protein inthe immunoprecipitation experiment shown in Example 10, were examined,so that the binding properties of each antibody with FGF-23 and thespecificities of recognition sites were revealed.

2. Method for Detecting FGF-23

(1) Method for Quantitatively Detecting FGF-23 and the Metabolitesthereof in Biological Samples While Distinguishing the Two

In clinical test, precise measurement of FGF-23 having activity in abiological sample is required. However, fragmentation products resultingfrom the cleavage of the FGF-23 protein upon the production of FGF-23 orthe cleavage of the FGF-23 protein upon the preparation of bloodsamples, which we have discovered, may be factors that disturb themeasurement of FGF-23. In particular, we have shown that activity waslost by cleavage between the 179^(th) and the 180^(th) amino acidresidues represented by SEQ ID NO: 1. Therefore, to detect FGF-23 havingactivity in a biological sample, the sandwich ELISA method using acombination of an antibody recognizing a part of the amino acid sequencebetween 25^(th) and the 179^(th) amino acid residues represented by SEQID NO: 1 and an antibody recognizing a part of an amino acid sequencebetween the 180^(th) and the 251^(st) amino acid residues represented bySEQ ID NO: I is effective. This can be conducted by combining a 2C3Bantibody or a 2C5L antibody, the N-terminal side recognition antibodythat we have obtained, with a 3C1E antibody or a 1D6A antibody, theC-terminal side recognition antibody (FIG. 5). Furthermore, when a serumsample is used in preparation of a blood sample, the full-length FGF-23existing in blood may be cleaved at a position between the 196^(th) andthe 197^(th) amino acid residues, or the 198^(th) and the 199^(th) aminoacid residues. In this case, when an antibody recognizing a portion ofthe 197^(th) and the following amino acid residues on the C-terminalside is used, it becomes impossible to carry out measurement reflectingthe original quantity of the protein existing in blood. Since thiscleavage cannot be observed in plasma (Example 26), even when a serumsample is used, the use of the 3C1E antibody, which we have obtained andwhich recognizes a position between the 180^(th) and the 196^(th) aminoacid residues, as an antibody for recognizing the C-terminus makes itpossible to carry out measurement reflecting the original quantity ofthe protein existing in blood.

Moreover, upon preparation of a serum sample, thrombin was shown to bean enzyme cleaving FGF-23 (Example 17). Hence, it is also possible toavoid effects resulting from the cleavage of FGF-23 in detection byadding a thrombin inhibitor upon preparation of a serum sample, so as tosuppress most cleavages of FGF-23 accompanying the preparation of theserum sample. The thrombin inhibitor may be any of those that do notobstruct the detection of FGF-23, and is preferably hirudin.

Moreover, the present application relates to a kit for detecting FGF-23containing an antibody recognizing a part of the amino acid sequencebetween the 25^(th) and the 179^(th) amino acid residues represented bySEQ ID NO: 1 and an antibody recognizing a part of the amino acidsequence between the 180^(th) and the 251^(st) amino acid residuesrepresented by SEQ ID NO: 1. In addition to anti-FGF-23 antibodies, ifnecessary, a stabilizer, a pH adjuster, or the like may be contained inthe kit for detecting FGF-23 of the present application.

(2) Method for Detecting FGF-23 with High Sensitivity

Elucidation of the relationship between the in vivo action of FGF-23 andmorbidity is clinically useful and is useful in differential diagnosisof tumor-induced osteomalacia and diagnosis of hereditaryhypophosphatemic rickets (ADHR, XLH: X-linked hypophosphatemic rickets).Hypophosphatemia, rickets, and osteomalacia, for which no dystrophia andfamily history are confirmed, are diseases the causes of which areunable to be specified, and thus diagnoses therefor are currentlydifficult. When an elevated concentration of FGF-23 in blood is observedin such a patient, it is possible to draw up therapeutic guidelines byconfirming differential diagnosis of hereditary diseases based on theconfirmation of gene mutations, or by finding tumors using a detailedmethod for detecting tumors that makes it enable the treatment oftumor-induced osteomalacia. Furthermore, because of the possible deepinvolvement of FGF-23 in biofunctions as a factor controlling phosphorusmetabolism and/or a factor controlling vitamin D metabolism, it isthought that the blood concentration fluctuates in morbidity of diseasesaccompanied by abnormal mineral metabolism, diseases of renal functions,diseases of bone metabolism, and the like. Thus, comparison of theaverage concentration of FGF-23 in the blood of the healthy adult with aconcentration of FGF-23 in the blood of a patient with such a diseasemay be useful in understanding the morbidity, selecting a therapeuticmethod, and determining a therapeutic plan. To construct such a FGF-23detection system, detection sensitivity allowing the FGF-23concentrations in blood to be measured is required. We have examined bythe sandwich ELISA method various combinations of antibodies that wehave obtained, thereby enabling quantitative measurement of FGF-23concentrations in the blood of a healthy adult and a patient with such adisease.

Examples of a method for detecting a substance (referred to as anantigen molecule) that is recognized by an antibody utilizing anantibody include a method of collecting detectable quantities ofsubstrates utilizing the binding of antibodies with antigen molecules, amethod of detecting the presence of antigen molecules by detectingantibodies specifically binding to the antigen molecules to be detected,and a method of detecting the presence of antigen molecules by measuringcompetition that occurs when the antigen molecules to be detected areallowed to be present in specific binding of a known quantity ofsubstrates with antibodies. Examples of qualitative detection methodsutilizing these methods include Western blotting, an immunoprecipitationmethod, and an immunostaining method. Furthermore, examples ofquantitative measurement methods that are generally known includeradioimmunoassay, ELISA, FACS and the like. When modified or cleaved,antigen molecules to be recognized by antibodies will take a variety offorms. As a detection method that involves specifying a part of theseforms, combining antibodies that recognize different sites of a targetsubstance is effective. A representative example of this method issandwitch ELISA.

Upon the completion of the detection method of FGF-23 of the presentinvention, the presence of a plurality of molecular species resultingfrom cleavage of the FGF-23 protein is clearly shown in FIG. 1A. Inparticular, as shown in Example 16, it was shown that FGF-23 existing asa full-length protein in blood is cleaved in serum. As such, there hasbeen a need to develop a detection system considering such cleavage inorder to quantitatively and precisely detect FGF-23 in vivo. For thispurpose, the combined use of the above antibodies recognizing partialsequences of the FGF-23 protein has proven very useful. Regarding ELISAusing polyclonal antibodies, as shown in Example 7, it was shown thatthe FGF-23 protein and the cleaved fragments thereof can be selectivelymeasured. Moreover, regarding anti-FGF-23 monoclonal antibodies obtainedin Examples 3 and 4, specificities to the recognition sites of theFGF-23 protein were analyzed, so that it was revealed thatcharacteristics of binding with FGF-23 can be applied to sandwich ELISA.It was revealed that among the obtained antibodies, the 1D6A antibody,the 2C3B antibody, and the 3C1E antibody can be independently used asimmobilized antibodies, and can also be used as antibodies fordetection. On the other hand, the 2A2B antibody was improper for any ofthese uses. Furthermore, it was revealed that the FGF-23 protein iscleaved in vivo and upon the preparation of serum. By combiningantibodies respectively recognizing a sequence region between the25^(th) and the 179^(th) amino acids of the FGF-23 protein, a sequenceregion between the 180^(th) and the 196^(th) amino acids, and a sequenceregion between the 197^(th) and the 251^(st) amino acids represented bySEQ ID NO: 1, detection and measurement of the metabolites of the FGF-23protein can be realized. Among the antibodies of present invention, the2C3B antibody recognizes the sequence region between the 25^(th) and the179^(th) amino acids, and the 3C1E antibody recognizes the sequenceregion between the 180^(th) and the 196^(th) amino acids of the FGF-23protein represented by SEQ ID NO: 1. The 1D6A antibody recognizes thesequence region between the 237^(th) and the 251^(st) amino acids in SEQID NO: 1. In particular, to detect the FGF-23 protein having activityfor the purpose of clinical tests or the like, a combination of theantibody recognizing a sequence region between the 25^(th) and the179^(th) amino acids with the antibody recognizing the sequence regionbetween the 180^(th) and the 196^(th) amino acids is preferred in termsof cleavage that occurs upon the preparation of serum. Particularly, adetection system with high sensitivity was realized using the 2C3Bantibody or the 2C5L antibody as an immobilized antibody and the 3C1Eantibody as an antibody for detection. Moreover, to detect thefull-length FGF-23 protein, a combination of the antibody recognizingthe sequence region between the 25^(th) and the 179^(th) amino acidswith the antibody recognizing the sequence region between the 237^(th)and the 251^(st) amino acids is preferred. Particularly, a detectionsystem with high sensitivity was realized using the 2C3B antibody as animmobilized antibody and the 1D6A antibody as an antibody for detection.

3. Diagnosis Methods for Hypophosphatemic Diseases, Rickets, andOsteomalacia

It has been suggested that in ADHR patients, missense mutations of theFGF-23 gene are involved in the induction of hypophosphatemic diseases(The ADHR Consortium. Autosomal dominant hypophosphatemic rickets isassociated with mutations in FGF23. Nature Genet. 26: 345-348, 2000).Furthermore, we have identified FGF-23 as a factor inducing diseases,whereby tumors are produced in tumor-induced osteomalacia (Shimada T,Mizutani S, Muto T, Yoneya T, Hino R, Takeda S, Takeuchi Y, Fujita T,Fukumoto S, and Yamashita T. Cloning and characterization of FGF23 as acausative factor of tumor-induced osteomalacia. Proc Natl. Acad. Sci.98: 6500-6505, 2001). These studies have revealed the possibleinvolvement of FGF-23 in diseases accompanied by hypophosphatemicdiseases, rickets, or osteomalacia. However, there have been no methodsfor quantitatively analyzing FGF-23 in a living body. In the presentinvention, we have established a specific FGF-23 detection system asdescribed above. Using this system as shown in Example 19, FGF-23contained in blood samples collected from patients with tumor-inducedosteomalacia before tumorectomy and the same collected after tumorectomywere quantitatively measured. As shown in FIG. 14B, althoughsignificantly high blood FGF-23 levels were shown before operation,after tumorectomy, the blood FGF-23 concentrations decreased to near thedetection limit. In tumor-induced osteomalacia, tumors are generallysmall, and although symptoms such as hypophosphatemia, decreased muscleforce, ostealgia, or osteomalacia are exhibited, causative tumors maynot be found. In addition, when tumors are observed, it is impossible toconfirm that the tumor produces FGF-23, so that diagnosisdifferentiating between tumor-induced osteomalacia and otherhypophosphatemic diseases showing similar symptoms is difficult. Themeasurement of the present invention enables detection of increasedlevels of FGF-23 in blood in tumor-induced osteomalacia, and thusenables diagnosis of cases that have conventionally been impossible todiagnose.

It is said that XLH, which is a disease with the highest incidence rateamong patients with hereditary hypophosphatemia, is said to be presentin approximately 1 out of 20,000 people. The gene responsible for XLHhas been identified as PHEX. This gene comprises 22 exons, and the generegion spans 220 kb, so that it is currently impossible to analyzemutations causing this hereditary hypophosphatemia for the purpose ofdiagnosis. The presence of cases involving sudden onset and typescharacterized by onset in adults has been suggested. So far therelationship between PHEX and FGF-23 has not been clarified. As an XLHmodel animal, an Hyp mouse, which is a spontaneous mutant mouse, isknown. The lack of the 3′ region of the PHEX gene has been confirmed inthis mouse, and the mouse is known to develop hypophosphatemia, rickets,or osteomalacia. By the use of the measurement system of the presentinvention, as shown in Example 23, we measured blood concentrations ofFGF-23 in Hyp mice, and discovered that the FGF-23 concentrationstherein were significantly high.

Accordingly, it was shown that the antibody of the present invention,and the detection method or the measurement method using the antibody,enable diagnosis of tumor-induced osteomalacia and XLH. Moreover, it isconsidered that the measurement method of the present invention can beused for ADHR, which is another form of hypophosphatemic disease and maybe caused by mutations in FGF-23.

FGF-23 has activity of decreasing phosphorus concentrations and 1,25Dconcentrations in blood. However, the physiological role thereof has notyet been well elucidated. By conducting the later-described experimentof the neutralization of FGF-23 activity, we revealed that FGF-23 has animportant role in maintaining the metabolic balance of phosphorus or1,25D even under normal conditions. Therefore, FGF-23 may be involved inphosphorus metabolism, and morbidity of renal diseases, intestinaldiseases, mineral metabolic disorders, and diseases with abnormalvitamin D metabolism, with which phosphorus metabolism is deeplyinvolved. Furthermore, also in patients to which 1,25D or a derivativethereof has been administered, fluctuations in FGF-23 may affectmorbidity and therapeutic effects. The measurement system of the presentinvention is thought to make it possible to deepen the understanding ofthe morbidity of these diseases, and to implement more precise medicalpractice.

4. Therapeutic Methods Against Diseases, Characterized by SuppressingFGF-23 Activity

It is known in tumor-induced osteomalacia and ADHR that the excessiveaction of FGF-23 induces the diseases. Furthermore, in the presentinvention, we also revealed that in the case of XLH, high levels ofFGF-23 were exhibited in blood. Suppression of FGF-23 is thought to leadto improve hypophosphatemia, rickets, or osteomalacia. In terms of thefact that FGF-23 acts on epithelial cells of the proximal tubule of thekidney, the suppression of FGF-23 may be useful in treating kidneytubule dysfunction. Moreover, in terms of the role of FGF-23 incontrolling phosphorus metabolism and vitamin D metabolism, FGF-23 mayact so as to provide unfavorable effects in diseases involving abnormalphosphorus metabolism, diseases involving abnormal Ca metabolism,diseases involving abnormal bone metabolism, diseases involving abnormalmetabolism accompanying decreased renal functions, metabolicabnormalities accompanying hemodialysis conducted for renal failure, anddiseases accompanying kidney transplantation. Similarly, it has beenreported that hypophosphatemia is recognized at high frequencies afterkidney transplantation and the disease is accompanied by osteopenia,suggesting the involvement of FGF-23 also in these cases. In such cases,not only recovering normal levels of FGF-23 from abnormally elevatedlevels, but also further decreasing FGF-23 showing normal values aspharmacological treatment may be required. It is considered thatantibodies shown in Examples 27 and 28 capable of neutralizing ormodifying the activity of FGF-23 are useful in treating variousdiseases. As shown in Example 25, it was discovered that animalsexhibiting hyperphosphatemia due to decreased renal functions exhibitsignificantly high levels of FGF-23. It is considered that at least apart of abnormalities in vitamin D metabolism accompanying decreasedrenal functions is an effect resulting from the elevated levels ofFGF-23. It is also considered that diseases accompanying the abnormallyelevated levels of FGF-23 can be treated by neutralizing or removing theactivity of FGF-23 using the antibodies of the present invention. Inparticular, it is considered that since phosphorus metabolism, calciummetabolism, and vitamin D metabolism are corrected by the antibodies ofthe present invention, these antibodies may be useful against diseasesaccompanying abnormalities in mineral metabolism, such as hypocalcemia,hyperparathyroidism, ectopic calcification, and itching, or diseasesinvolving abnormalities in bone metabolism accompanying decreased renalfunctions, such as renal osteodystrophy, and dialysis osteopathy.Moreover, the use of the antibodies of the present invention may also beuseful against cardiovascular hypofunction accompanied by calcification,which is a problem in hemodialysis patients. Moreover, as shown inExample 27, it was revealed that even under normal metabolic conditions,FGF-23 has an important role in mineral metabolism and vitamin Dmetabolism. In view of not only the above fact that FGF-23 is induced inhyperphosphatemia, but also the facts that FGF-23 rapidly decreases1,25D and FGF-23 is rapidly induced by 1,25D, the antibodies of thepresent invention capable of neutralizing or modifying the activity ofFGF-23 may have extensive usefulness against diseases with abnormalitiesin mineral metabolism. This is because the antibodies not only controlphosphorus metabolism, vitamin D metabolism, and calcium metabolism bysuppressing the action of FGF-23, but also indirectly control hormonesfor calcium metabolism, such as parathyroid hormones and calcitonin. Inparticular, the antibodies of the present invention are expected to beeffective for treating osteoporosis, osteopenia, osteosclerosis, orPaget's disease exhibiting various forms of morbiditity in associationwith the balance in mineral metabolism and metabolic turnover, orhypercalcemia or hypoparathyroidism. As described above, the antibodiesof the present invention may be useful against one or more of thediseases described above.

5. Antibody for Neutralizing the Biological Activities of FGF-23

FGF-23 is thought to have an important role in metabolic control in aliving body, as described above. A method for controlling the biologicalactivities of such a factor is thought to be useful for therapiesagainst and prevention of various diseases. An antibody is characterizedby specific binding with an antigen molecule, and is known to have aneffect on the structure and functions of an antigen molecule, dependingon its recognition site. Thus, we aimed at isolation and identificationof an antibody capable of controlling the functions of FGF-23, andparticularly, capable of suppressing the biological activities ofFGF-23. As shown in Examples 27 and 28, we have discovered thatadministration of an antibody recognizing FGF-23 causes increases inserum phosphorus and in serum 1,25D concentrations. In particular, theantibodies of the present invention used in these examples caused thecomplete disappearance of the activity of human FGF-23 to lower serumphosphorus and 1,25D, which had been experimentally produced in vivo inmice. Furthermore, in control mice that had not been caused to producehuman FGF-23, elevated levels of serum phosphorus and vitamin D wereconfirmed. This phenomenon was completely opposite to the changesobserved when FGF-23 had been administered. Thus, the antibody of thepresent invention is thought also to suppress mouse endogenous FGF-23.Furthermore, this phenomenon shows that FGF-23 functions as a factorcontrolling phosphorus metabolism and vitamin D metabolism not only in astate of morbidity, but also in a normal state. The antibodies of thepresent invention capable of causing the disappearance or attenuation ofthe biological activity of FGF-23 can control physiological andpathological conditions that are the reflection of the biologicalactivity of FGF-23. The range of the biological activity of FGF-23 thatthe antibody of the present invention can control is not limited only tophosphorus metabolism and vitamin D metabolism. The antibody can controlevery biological activity and physiological activity of FGF-23.

Moreover, two or more types of antibodies recognizing different sites ofFGF-23 as shown in Example 31, that is, different epitopes, may be used.In this case, the neutralization activity of the antibodies is enhanced,so that the action time of the antibodies can also be maintained for along period of time. Examples of a combination of 2 or more types ofantibodies include an antibody recognizing an amino acid sequencebetween the 180^(th) and the 194^(th) or between the 237^(th) and the251^(st) amino acid residues represented by SEQ ID NO: 1, and anantibody produced by a hybridoma whose accession number is FERM BP-7838,FERM BP-7839, FERM BP-7840 or FERM BP-8268.

6. Pharmaceutical Composition Comprising Anti-FGF-23 Antibody

The antibody or the pharmaceutical composition of the present inventioncan be applied for therapies or prevention of various diseases orsymptoms in which FGF-23 produced in vivo or in cells expressing FGF-23is involved. The antibody of the present invention can be used as apharmaceutical composition against tumor-induced osteomalacia, ADHR, andXLH, and can be expected to have an effect of improving the conditions,which are observed in common among these diseases, of hypoposphatemia,failure of bone calcification, ostealgia, decreased muscle force,skeletal deformation, growth disease, and hypo-1,25D-hemia (diseasecharacterized by low levels of 1,25D in blood). As described above,FGF-23 plays an important role in physiological conditions. The antibodyof the present invention can be used as a pharmaceutical compositiontherapeutically and prophylactically against diseases resulting fromabnormalities in mineral metabolism or vitamin D metabolism such asosteoporosis, rickets, hypercalcemia, hypocalcemia, ectopiccalcification, osteosclerosis, disorders of bone extension, disorders ofbone calcification, Paget's disease, hyperparathyroidism,hypoparathyroidism, and itching, by controlling the phosphorusmetabolism-controlling action of FGF-23, or thecalcium-metabolism-controlling action mediated by vitamin D metabolismthat is controlled by FGF-23. Furthermore, it is clear that blood FGF-23concentrations increase in renal failure. Hence, the antibody of thepresent invention can be used therapeutically and prophylactically as apharmaceutical composition against complications of renal failure orblood dialysis, represented by renal osteodystrophy, dialyticosteopathy, dysfunction of renal tubules, and the like.

Moreover, the antibody of the present invention bound with a therapeuticreagent can be used as a pharmaceutical composition. In tumor-inducedosteomalacia, it is known that a tumor excessively produces FGF-23 so asto induce morbidity. Currently, a sole therapeutic method involves theremoval of the causative tumor. However, a causative tumor is oftensmall. There have been many case reports of causative tumor beingfinally discovered by energetic MRI search. In addition, there may bemany cases where a diagnosis of hypophosphatemia or osteomalacia withunknown causes is made, because no tumors could be found. The antibodyof the present invention is considered to be accumulated inFGF-23-producing tumors in such diseases, because of affinity forFGF-23. As a method for degenerating tumors using this property, it iseffective to use a therapeutic reagent bound to the antibody of thepresent invention. Examples of a therapeutic reagent to be bound to theantibody include (1) radiation isotopes such as iodo (131 Iodine: 131I;125 Iodine: 125I), yttrium (90Yttrium: 90Y), indium (111 Indium: 111In),and technetium (99m Technetium: 99mTc) (J. W. Goding, MonoclonalAntibodies: principles and practice, 1993 ACADEMIC PRESS), (2) bacterialtoxins such as Pseudomonas toxin (Pseudomonas exotoxin), diphtheriatoxin, and ricin, and (3) chemotherapeutants such as methotrexate,mitomycin, and calicheamicin (D. J. King, Applications and Engineeringof Monoclonal Antibodies, 1998 T. J. International Ltd, M. L.Grossbard., Monoclonal Antibody-Based Therapy of Cancer., 1998 MarcelDekker Inc). More preferably, a prodrug such as Maytansinoid ispreferred (Chari et al., Cancer Res., 1992 Vol 52: 127, Liu et al., ProcNatl Acad Sci USA., 1996 Vol93:8681).

In addition, the scope of the present invention also encompasses apharmaceutical preparation comprising a purified product of theanti-human FGF-23 antibody. Such a pharmaceutical preparation preferablycontains a physiologically acceptable diluent or carrier in addition toan antibody, and may be a product mixed with other antibodies or otherdrugs such as antibiotics. Examples of an appropriate carrier include,but are not limited to, physiological saline, phosphate buffered saline,phosphate buffered saline glucose solution, and buffered physiologicalsaline. Alternatively, antibodies may be frozen and dried (freeze-dry),and the above buffered aqueous solution may be added to the antibodies,if necessary, so as to reconstitute and use the antibodies. The route ofadministration may be oral or non-enteral, including intravenous,intramuscular, subcutaneous, and intraperitoneal injections or drugdelivery.

When the pharmaceutical composition of the present invention isadministered to a patient, the effective dosage per administration isselected from the range between 20 ng and 200 mg per kg of body weight.Alternatively, a dosage of 0.001 to 10000 mg/body weight, preferably0.005 to 2000 mg/body weight, and more preferably 0.01 to 1000 mg/bodyweight per patient can be selected. However, the dosage of thepharmaceutical composition of the present invention is not limited tothese dosages.

7. Medical Appliances Containing Anti-FGF-23 Antibody

In therapeutic techniques for hemodialysis, plasma exchange, and cellcollection, substances in a living body are removed, exchanged, andcollected from a part of collected body fluids or body fluids subjectedto extracorporal circulation. In such therapeutic techniques, theantibody of the present invention is useful in selective removal ofFGF-23 molecules in a living body and selective collection of cellsexpressing FGF-23, utilizing the property of specifically binding withFGF-23 molecules. In hemodialysis and plasma exchange, a potentialmethod involves the antibody of the present invention being immobilizedto parts of materials with which body fluids come into contact. Asmaterials of a dialysis membrane, in addition to a cellulose membrane, asynthetic polymer membrane such as a polyacrylonitrile membrane, apolymethylmethacrytate membrane, an ethylene vinyl alcohol membrane, apolysulfone membrane, a polyamide membrane, apolyethersulfone/polyarylate membrane, and the like are used. Such amembrane to which the antibodies are immobilized by covalent binding canbe used for hemodialysis. Furthermore, there may be a method forseparating body fluids using a column filled with beads, such assepharose beads, to which the antibodies have been bound. In addition,another possible method involves immobilizing the antibodies on magneticbeads, admixing the antibodies with antibody-binding molecules topromote binding, and then collecting complexes of the antibodies andtarget substances using a magnet. Cells are collected by such a methodand used for therapies. As described above, the antibody of the presentinvention that is bound to a base material that is appropriate as amedical device can be used as a medical appliance.

8. Method for Controlling the Molecular Structure and BiologicalActivity of FGF-23

An important point for maintaining the biological activity of a proteinin vivo is to allow the protein to exist while maintaining thethree-dimensional structure thereof in vivo. Among biofactors, asobserved in the case of an insulin-like growth factor (IGF) or atransforming growth-factor β (TGF-β), there are many cases where abiofactor binds in vivo to another protein so as to control biologicalactivity. For FGF-23, no binding protein is known to date. Examples ofthe antibody of the present invention include the 2C3B antibody that isthought to strongly recognize the structure of FGF-23 without binding toa partial peptide as shown in Example 9. It is conceivable to be able tocontrol the biological activity of FGF-23 in vivo by creating a statewhere these antibodies or parts of these antibodies are bound to FGF-23.

9. Method for Efficiently Removing FGF-23

Significantly decreased renal functions require the removal of uremicsubstances from the body. For the removal of uremic substances with lowmolecular weights, hemodialysis using a dialytic membrane is practicallyused. However, the removal of protein with high molecular weights isstill a problem. The antibody of the present invention can be used forthe specific removal of FGF-23. As performed in hemodialysis, blood forextracorporeal circulation is brought into contact with a material towhich the antibody of the present invention has been immobilized, sothat FGF-23 can be selectively removed. The use of the antibody as atherapeutic tool is possible for diseases where FGF-23 existsexcessively. As shown in Example 25, based on the fact that high levelsof FGF-23 are shown at the time of renal failure, FGF-23 may be a factorinducing dialysis complications. In particular, since FGF-23 has actionto lower 1, 25D, there is a high probability that FGF-23 functions as afactor inducing a decrease in 1,25D accompanying decreased renalfunctions. Thus, a method for removing FGF-23 using the antibody of thepresent invention in dialysis patients may be useful in therapy fordialysis complications.

As described above, we have completed the present invention by obtainingan antibody that can not only recognize FGF-23, but also controlphysiological, pharmacological, and pathological actions in which FGF-23is involved, and can be applied for treating, preventing, and diagnosingdiseases.

10. Specification of Competitive Antibody

Upon binding with FGF-23, an antibody that recognizes the same site asthat of the antibody of the present invention or a site very close tosuch site is considered to exhibit properties equivalent to thecharacteristics shown herein. Such a substantially equivalent antibodycan be distinguished from other antibodies by conducting experimentsrelated to competitiveness. When 2 or more types of antibodies areallowed to coexist, antibodies showing the property of bindingexclusively to each other upon binding with antigens are defined ascompetitive antibodies. To specify the competitive antibody of thepresent invention, under conditions where a labeled antibody of theinvention binds to the FGF-23 protein, unlabeled antibodies are allowedto exist in excessive quantities, so that determination can beperformed. When added antibodies significantly lower the binding of theantibodies of the invention with the FGF-23 protein, it can bedetermined that the added antibodies compete with the antibodies of theinvention.

The specification includes part or all of the contents as disclosed inthe specifications and/or drawings of Japanese Patent Application Nos.2001-401689 and 2002-262020, which are priority documents of the presentapplication.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows recombinant FGF-23 protein and the metabolites thereof asdetected by Western blotting using anti-FGF-23 polyclonal antibodiesafter the separation of the culture supernatant of CHO-FGF23H cells bySDS-polyacrylamide gel electrophoresis. In the culture supernatant, thefull-length FGF-23H protein, and the N-terminal fragment and theC-terminal fragment generated by cleavage between the 179^(th) and the180^(th) amino acid residues in the amino acid sequence of SEQ ID NO: 1,are present. The full-length FGF-23H protein and the N-terminal fragmentpeptide were recognized using hFGF23-48 antibodies and hFGF23-148antibodies. As N-terminal fragment, the presence of small fragmentedpeptides was observed. The full-length protein and the C-terminalfragment were detected using anti-His6-tag antibodies. (Example 2)

FIG. 1B shows the FGF-23 full-length protein, the N-terminal fragmentand the C-terminal fragment purified from the culture supernatant ofCHO-FGF23 cells as detected by CBB staining after separation bySDS-polyacrylamide gel electrophoresis. (Example 2)

FIG. 1C shows a recombinant FGF-23 protein, a recombinant FGF-23RQprotein, and the metabolite thereof as detected by Western blottingusing an anti-FGF 23-148 antibody after separately obtaining the culturesupernatants of CHO-FGF23H and CHO-FGF23RQ cells, and then separatingthe cells by SDS-polyacrylamide gel electrophoresis. (Example 5)

FIG. 2 shows the FGF-23H protein as measured by sandwich ELISA using animmobilized hFGF23-25 antibody, and an hFGF23-237 antibody as antibodiesfor detection, which are anti-FGF-23 polyclonal antibodies. (Example 7)

FIG. 3 shows the results as detected using 2A2B and 1C3H antibodiesafter immunoprecipitating the FGF-23H protein using anti-FGF-23monoclonal antibodies, and separating the protein by SDS-polyacrylamidegel electrophoresis. (Example 10)

FIG. 4 shows the purified FGF-23H full-length protein, the N-terminalfragment polypeptide, and the C-terminal fragment polypeptide asdetected by sandwich ELISA using immobilized anti-FGF-23 monoclonalantibodies and anti-FGF-23 polyclonal antibodies as antibodies fordetection. (Example 11)

FIG. 5 schematically shows the full-length FGF-23 protein and thecleavage sites thereof, and the recognition sites of the anti-FGF-23monoclonal antibodies. (Example 12)

FIG. 6 shows the purified FGF-23 protein as quantitatively measured bythe ELISA system using the 2C3B antibody as an immobilized antibody andthe 1C3H, 1D6A, and 3C1E antibodies as antibodies for detection.(Example 13)

FIG. 7 shows the results as detected using the 2A2B and 1C3H antibodiesafter collecting by an immunoprecipitation method the FGF-23H proteincontained in serum collected from nude mice having CHO-FGF23H cellstransplanted therein using resins onto which the 1C3H, 1D6A, and 2C3Bantibodies were immobilized, and then separating the protein bySDS-polyacrylamide gel electrophoresis. (Example 15)

FIG. 8 shows the results as detected by Western blotting using the 2A2Bantibody after adding rat serum to 20 ng of purified FGF-23 protein toresult in a final concentration of 50%, admixing the resultant, and thenseparating metabolites generated by 0, 0.5, 1, 2, 4, 8, or 24 hours ofincubation by SDS-polyacrylamide gel electrophoresis. (Example 16)

FIG. 9 shows metabolites derived from the purified FGF-23 protein asdetected by Western blotting using the 2A2B antibody after adding humanserum or human plasma to 20 ng of the purified FGF-23 protein to resultin a final concentration of 50%, performing incubation at 37° C. for 3hours, and performing separation by SDS-polyacrylamide gelelectrophoresis. (Example 16)

FIG. 10 shows metabolites derived from the purified FGF-23 protein asdetected by Western blotting using the 2A2B antibody after addingthrombin to 20 ng of purified FGF-23 protein at a final concentration of1 unit/ml, performing incubation for 3 hours, and then subjecting thesolution to SDS-polyacrylamide gel electrophoresis for separation.(Example 17)

FIG. 11 shows metabolites derived from the purified FGF-23 protein asdetected by Western blotting using the 2A2B antibody after adding ratserum to 20 ng of purified FGF-23 protein in the presence or the absenceof hirudine, a thrombin selective inhibitor, at a final concentration of50%, performing incubation for 4 hours, and then performing separationby SDS-polyacrylamide gel electrophoresis. (Example 17)

FIG. 12 shows a polypeptide of 22 kDa as identified by CBB stainingafter admixing 10 μg of purified FGF-23 protein and rat serum for 24hours to generate the polypeptide of 22 kDa, purifying the polypeptideof 22 kDa using an anti-2A2B antibody column, and then performingseparation by SDS-polyacrylamide gel electrophoresis. (Example 18)

FIG. 13 shows the FGF-23 protein in blood samples collected before andafter extraction of causative tumors of patients with neoplasticosteomalacia, as measured by the ELISA system whereby detection wasperformed using the 2C3B antibody as an immobilized antibody, and the3C1E or the 1D6A antibody as an antibody for detection. (Example 19)

FIG. 14 shows the results of quantifying FGF-23 protein in the bloodsamples collected before and after extraction of causative tumors ofpatients with neoplastic osteomalacia. (Examples 19)

FIG. 15 shows the results of detecting the presence of FGF-23 protein intumor tissues extracted from patients with neoplastic osteomalacia byimmunohistological staining. (Example 20)

FIG. 16A shows mouse FGF-23RQ protein as detected by Western blottingusing the 3C1E antibody, which was an anti-human FGF-23 monoclonalantibody, after separating purified mouse FGF-23RQ protein bySDS-polyacrylamide gel electrophoresis. (Example 22)

FIG. 16B shows a mouse FGF-23 protein solution as measured using the2C3B antibody as an immobilized antibody and the 3C1E antibody as anantibody for detection after serial dilution of purified mouse FGF-23protein. (Example 22)

FIG. 17 shows the results of measuring endogenous FGF-23 protein inserum collected from 27- to 30-week-old Hyp mice and control mice usingthe ELISA system using the 2C3B antibody as an immobilized antibody, andthe 3C1E antibody as an antibody for detection. (Example 23)

FIG. 18A shows serum 1,25D concentrations as measured afteradministering 5 μg of purified FGF-23H protein per mouse to six6-week-old BALB/c male mice, and then collecting blood at 1, 4, and 9hours after administration. (Example 24)

FIG. 18B shows serum FGF-23 quantities as measured after administering0.025 μg of 1,25D intraperitoneally to six 7-week-old BALB/c male mice,and then collecting blood at 8 hours after administration. (Example 24)

FIG. 19A shows serum phosphate concentrations measured after feedingCE-2 (CLEA JAPAN, INC.), with which adenine had been mixed at a rate of0.75%, to 7-week-old Wistar rats to produce a renal failurehyperphosphatemia model, feeding CE-2 to a control group, and thencollecting blood 3 weeks after administration of feed mixed withadenine. (Example 25)

FIG. 19B shows serum and urine creatinine concentrations measured afterfeeding CE-2 (CLEA JAPAN, INC.), with which adenine had been mixed at arate of 0.75%, to 7-week-old Wistar rats to produce a renal failurehyperphosphatemia model, feeding CE-2 to a control group, and collectingblood 3 weeks after administration of feed mixed with adenine and urinefor 24 hours from the rats housed in metabolism cages. (Example 25)

FIG. 19C shows serum FGF-23 concentrations as measured using the ELISAsystem using the 2C3B antibody as an immobilized antibody, and the 3C1Eantibody as an antibody for detection after feeding CE-2 (CLEA JAPAN,INC.), with which adenine had been mixed at a rate of 0.75%, to7-week-old Wistar rats to produce a renal failure hyperphosphatemiamodel, feeding CE-2 to a control group, and then collecting blood 3weeks after administration of feed mixed with adenine. (Example 25)

FIG. 20 shows the results of detecting FGF-23 protein existing in theplasma of patients with neoplastic osteomalacia by animmunoprecipitation method. (Example 26)

FIG. 21A shows the concentrations of serum phosphate, calcium, and1,25-dihydroxy vitamin D at 24 hours after administration of variousmonoclonal antibodies and a vehicle. (Example 27)

FIG. 21B shows serum 1,25-dihydroxy vitamin D concentrations 8 hoursafter the administration of the 3C1E monoclonal antibody and a vehicle.(Example 28)

FIG. 22A shows serum 1,25-dihydroxy vitamin D concentrations on day 7after the start of keeping in a group (adenine) to which feed mixed withadenine had been given, or a group (control) to which general feed hadbeen given. Measured values are expressed with average value±standarddeviation. “*” and “**” indicate p<0.05 and p<0.01, respectively, whichare the results of tests of significance conducted by Student-t.(Example 29)

FIG. 22B shows serum FGF-23 concentrations on day 7 after the start ofkeeping in a group (adenine) to which feed mixed with adenine had beengiven, or in a group (control) to which general feed had been given.Measured values are denoted with average value±standard deviation. “*”and “**” indicate p<0.05 and p<0.01, respectively, which are the resultsof tests of significance conducted by Student-t. (Example 29)

FIG. 22C is a correlation diagram wherein the serum FGF-23concentrations and 1,25-dihydroxy vitamin D concentrations on day 7after the start of keeping in a group to which feed mixed with adeninehad been given are plotted by individual mice. (Example 29)

FIG. 22D shows changes in serum 1,25-dihydroxy vitamin D concentrationsat 12 hours after administering the 3C1E antibody or a vehicle on day 7after the start of keeping to a group (adenine) to which feed mixed withadenine had been given, or a group (control) to which general feed hadbeen given. Measured values are expressed with average value±standarddeviation. “*” and “**” indicate p<0.05 and p<0.01, respectively, whichare the results of tests of significance conducted by Student-t.(Example 29)

FIG. 23 shows the gender, identified mutation sites of a phex gene, ageupon blood collection, and serum FGF-23 concentrations of 6 patientswith X-linked hypophosphatemic rickets (XLH). (Example 30)

FIG. 24 shows serum phosphate concentrations on day 1 and day 2 afteradministering the mixture of 2 antibodies (2C3B and 3C1E antibodies) ora vehicle (PBS) to normal mice. Measured values are expressed withaverage value±standard deviation. “*” and “**” indicate p<0.05 andp<0.01, respectively, which are the results of tests of significanceconducted by Student-t, compared with the group to which vehicles hadbeen administered. “a” and “b” indicate p<0.05 and p<0.01, respectively,which are the results of tests of significance conducted by Student-t.(Example 31)

FIG. 25A shows blood phosphate concentrations, 1,25-dihydroxy vitamin Dconcentrations, and total alkaline phosphatase activity on days 1 and 7after repeatedly administering the mixture of antibodies (Ab), comprisedof the 2C3B antibody and the 3C1E antibody, or a vehicle (PBS) towild-type mice (WT) and Hyp mice (Hyp). Blood phosphorus concentrationsand alkaline phosphatase activity values are expressed with averagevalue±standard deviation. “*” and “**” indicate p<0.01 and p<0.001,respectively, which are the results of tests of significance conductedby Student-t. Blood 1,25-dihydroxy vitamin D concentration was measuredusing plasma prepared by admixing an equivalent volume of plasma samplescollected from each group. (Example 32)

FIG. 25B shows blood phosphate concentrations and 1,25-dihydroxy vitaminD concentrations on day 4 after single administration of the mixture ofantibodies (Ab), comprised of the 2C3B antibody and the 3C1E antibody,or a vehicle (PBS) to wild-type mice (WT) and Hyp mice (Hyp). Resultsare expressed with average value±standard deviation. “*” and “**”indicate p<0.01 and p<0.001, respectively, which are the results oftests of significance conducted by Student-t. (Example 32)

FIG. 25C shows the expression amount (upper case) of NaPi2a protein inBBMV prepared from the kidney as analyzed by the Western blottingmethod, and phosphate transport activity (lower case) thereof on day 4after single administration of the mixture of antibodies (Ab), the 2C3Bantibody and the 3C1E antibody, or a vehicle (PBS) was performed forwild-type mice (WT) and Hyp mice (Hyp). To correct the protein levels ofBBMV subjected to the test, the blot used for Western blotting wassubjected to CBB staining, and an image of stained beta-actin is shownat the same time. Results of measuring phosphate transport activity areexpressed with average value±standard deviation and n=3 for each case.(Example 32)

FIG. 25D shows changes in the expression of a 1αOHase gene as analyzedby Northern blotting using RNA prepared from the kidney on day 4 aftersingle administration of the mixture of antibodies (Ab), the 2C3Bantibody and the 3C1E antibody, or a vehicle (PBS) was performed forwild-type mice (WT) and Hyp mice (Hyp). (Example 32)

FIG. 26 shows changes with time in blood osteocalcin concentrations whenthe 3C1E antibody or a vehicle was administered to normal rats. Resultsare expressed with average value±standard deviation. “*” and “**”indicate p<0.01 and p<0.001, respectively, which are the results oftests of significance conducted by Student-t. (Example 33)

FIG. 27 shows magnified X-ray images of femora, tibiae, and costaeextracted after administration of the mixture of antibodies (Ab),comprised of the 2C3B antibody and the 3C1E antibody, or a vehicle (PBS)to wild-type mice (WT) and Hyp mice (Hyp). (Example 32)

FIG. 28 shows comparison of images of bone tissues of the proximal partof the tibia and the distal part of the femur of Hyp mice (Hyp/antibody)to which the mixture of antibodies (the 2C3B antibody and the 3C1Eantibody) was repeatedly administered, with images of the tissues of therelevant regions of Hyp (Hyp/vehicle) and wild-type mice(Wild-type/vehicle) to which vehicles were administered. The extractedtibiae and femora were subjected to Villanueva Bone staining. They wereresin-embedded, and then prepared to result in 5 μm-thicknondecalcification sections. These samples were stained differently:osteoid is purple, calcified bone is light orange, low-calcified bone islight brown, and embedded cells are light purple under visible light.(Example 32)

FIG. 29A shows blood osteocalcin concentrations (IRMA kit, Immutopics,Inc.) on week 2 after ovariectomy of established groups: a group(sham/PBS) of mice subjected to sham operation to which vehicles (PBS)were administered, a group (OVX/Abs) of mice subjected to ovariectomy towhich the mixture of antibodies, comprised of the 2C3B antibody and the3C1E antibody, were administered, or a group (OVX/PBS) of mice subjectedto ovariectomy to which vehicles (PBS) were administered. Bloodosteocalcin concentrations are expressed with average value±standarderror. “*” and “**” indicate p<0.01, and p<0.001, respectively, whichare the results of tests of significance conducted by Student-t.(Example 34)

FIG. 29B shows blood osteocalcin concentrations (IRMA kit, Immutopics,Inc.) on week 4 after ovariectomy of established groups: a group(sham/PBS) of mice subjected to sham operation to which vehicles (PBS)were administered, a group (OVX/Abs) of mice subjected to ovariectomy towhich the mixture of antibodies, comprised of the 2C3B antibody and the3C1E antibody, were administered, or a group (OVX/PBS) of mice subjectedto ovariectomy to which vehicles (PBS) were administered. Bloodosteocalcin concentrations are expressed with average value±standarderror. “*” and “**” indicate p<0.01, and p<0.001, respectively, whichare the results of tests of significance were conducted by Student-t.(Example 34)

FIG. 30A shows bone-salt quantities in the femora on week 4 afterovariectomy of established groups: a group (sham/PBS) of mice subjectedto sham operation to which a vehicle (PBS) was administered, a group(OVX/Abs) of mice subjected to ovariectomy to which the mixture ofantibodies comprised of the 2C3B antibody and the 3C1E antibody, wereadministered, or a group (OVX/PBS) of mice subjected to ovariectomy towhich a vehicle (PBS) was administered. Bone-salt quantities and bonedensities of the femora are expressed with average value±standard error.“*” and “**” indicate p<0.05 and p<0.01, respectively, which are theresults of tests of significance conducted by Student-t. (Example 34)

FIG. 30B shows bone densities (Bone Densitometer, Model DCS-600, ALOKACO., LTD.) on week 4 after ovariectomy of established groups: a group(sham/PBS) of mice subjected to sham operation to which a vehicle (PBS)was administered, a group (OVX/Abs) of mice subjected to ovariectomy towhich the mixture of antibodies, comprised of the 2C3B antibody and the3C1E antibody, were administered, or a group (OVX/PBS) of mice subjectedto ovariectomy to which a vehicle (PBS) was administered. Bone-saltquantities and bone densities of the femora are expressed with averagevalue±standard error. “*” and “**” indicate p<0.05 and p<0.01,respectively, which are the results of tests of significance conductedby Student-t. (Example 34)

FIG. 31A shows the full-lengths of caudal vertebra as periodicallymeasured after repeated subcutaneous administration (once a week) ofvehicles to Hyp mice (Hyp/vehicle), the mixture of antibodies, comprisedof the 2C3B antibody and the 3C1E antibody, at 4 mg/kg to Hyp mice(Hyp/low dose of antibodies), the same mixture at 16 mg/kg to Hyp mice(Hyp/high dose of antibodies), or vehicles to wild-type mice(wild-type/vehicle). Results are expressed with average value±standarddeviation. “*” and “**” indicate p<0.05 and p<0.01, respectively, whichare the results of tests of significance conducted by Student-t, whencompared with the Hyp group to which vehicles were administered.(Example 35)

FIG. 31B shows the lengths of tibia as periodically measured afterrepeated subcutaneous administration (once a week) of a vehicle to Hypmice (Hyp/vehicle), the mixture of antibodies, comprised of the 2C3Bantibody and the 3C1E antibody, at 4 mg/kg to Hyp mice (Hyp/low dose ofantibodies), or the same mixture at 16 mg/kg to Hyp mice (Hyp/high doseof antibodies), and a vehicle to wild-type mice (wild-type/vehicle).Results are expressed with average value±standard deviation. “**”indicates p<0.01 which is the result of a test of significant conductedby Student-t, when compared with the Hyp group to which a vehicle wasadministered. (Example 35)

FIG. 31C shows changes in body weight as periodically measured afterrepeated subcutaneous administration (once a week) of a vehicle to Hypmice (Hyp/vehicle), the mixture of antibodies, comprised of the 2C3Bantibody and the 3C1E antibody, at 4 mg/kg to Hyp mice (Hyp/low dose ofantibodies), or the same mixture at 16 mg/kg to Hyp mice (Hyp/high doseof antibodies), and a vehicle to wild-type mice (wild-type/vehicle).Results are expressed with average value±standard deviation. “*” and“**” indicate p<0.05, and p<0.01, respectively, which are the results oftests of significance conducted by Student-t, when compared with the Hypgroup to which a vehicle were administered. (Example 35)

FIG. 32 shows the proportions of bone ash weight in dry bone weight whenthe femora were extracted and incinerated on day 31 after repeatedsubcutaneous administration (once a week) of a vehicle to Hyp mice(Hyp/vehicle), the mixture of antibodies, comprised of the 2C3B antibodyand the 3C1E antibody, at 4 mg/kg to Hyp mice (Hyp/low dose ofantibodies), or the same mixture of antibodies at 16 mg/kg to Hyp mice(Hyp/high dose of antibodies), and a vehicle to wild-type mice(wild-type/vehicle). Results are expressed with average value±standarddeviation. “*” indicates p<0.001 which is the result of a test ofsignificant conducted by Student-t, when compared with the Hyp group towhich a vehicle was administered.

FIG. 33 shows the results of quantitatively measuring purified FGF-23protein by the ELISA system using the 2C3B or the 2C5L antibody as animmobilized antibody, and the 3C1E antibody as an antibody fordetection. (Example 36)

FIG. 34 shows blood phosphate concentrations before administration andat 24 hours after administration of a vehicle to a group(untreated/vehicle) of untreated normal mice, and a vehicle(FGF-23/PBS), the 2C5L antibody (FGF-23/2C5L), or the 2C3B antibody(FGF-23/2C3B) to each group that had been continuously administered withhuman recombinant FGF-23 using an osmotic pump. Results are expressedwith average value±standard deviation. “*” indicates p<0.001 which isthe result of a test of significant conducted by Student-t for eachgroup before antibody administration, when compared with a group towhich a vehicle was administered. “#” and “##” indicate p<0.01 andp<0.001, respectively, which are the results of tests conducted byStudent-t for each group before and at 24 hours after administration ofthe antibodies. “+”, “++,” and “+++” indicate p<0.05, p<0.01, andp<0.001, respectively, which are the results of tests for significantconducted by Student-t for each group after administration of theantibodies, when compared with those of the group 24 hours afteradministration of FGF-23/PBS. (Example 38)

SEQUENCE LISTING FREE TEXT

SEQ ID NOs: 2-8: synthetic DNA

SEQ ID NOs: 9-24: synthetic peptide

SEQ ID NOs: 25-36: synthetic DNA

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained more specifically with referenceto examples, but the present invention is not limited to the embodimentor technical scope described in the examples.

EXAMPLE 1 Construction of Recombinant FGF-23 Expression Vector

(1) Preparation of FGF-23H Protein Expression Vector

cDNA encoding FGF-23 was amplified after keeping the temperature at 96°C. for 1 minute by performing 35 cycles of PCR process, each cycleconsisting of 96° C. for 30 seconds, 55° C. for 30 seconds, and 72° C.for 30 seconds using a cDNA library of a responsible tumor of neoplasticosteomalacia as a template, and a FlEcoRI primer (SEQ ID NO: 2), aLHisNot primer (SEQ ID NO: 3), and LA-Taq DNA polymerase. The FlEcoRIprimer was annealed to a sequence that is present further upstream onthe 5′ side of a nucleotide sequence encoding FGF-23, so as to add anEcoR I restriction enzyme site to the 5′ side of a region of theamplification fragment encoding FGF-23. The LHisNot primer contains asequence annealing with a sequence on the 5 side of the terminationcodon of a sequence encoding FGF-23, and a sequence encoding a His6-tagsequence (His-His-His-His-His-His), followed by the termination codonand a Not I restriction enzyme sequence. As a result, the amplificationfragment encoded a sequence of the FGF-23 protein having the His6-tagsequence added to the C-terminus and having the Not I restriction enzymesite located downstream of the His6-tag sequence. This amplificationfragment was digested with EcoR I and Not I, and then ligated to apcDNA3.1Zeo vector (Invitrogen, U.S.A.), which was an animal cellexpression vector that had been digested in a similar manner as EcoR Iand Not I. The thus prepared expression vector was cloned, and thenucleotide sequence was determined, thereby confirming that it encodedthe target FGF-23 protein having the His6-tag sequence added thereto.This vector is referred to as pcDNAFGF-23H. FlEcoRICCGGAATTCAGCCACTCAGAGCAGGGCACG (SEQ ID NO: 2) LHisNot:ATAAGAATGCGGCCGCTCAATGGTGATGGTGATGAT (SEQ ID NO: 3) GGATGAACTTGGCGAA(2) Construction of FGF-23 Protein Expression Vector

Amplification was performed after keeping the temperature at 94° C. for1 minute, and then performing 25 cycles of PCR process, each cycleconsisting of 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C.for 1 minute using pcDNA/FGF-23H as a template, and a FlEcoRI primer,LNot primer (SEQ ID NO: 4), and LA-Taq DNA polymerase. After reaction, acDNA fragment encoding the FGF-23 protein was prepared by blunt-endingthe termini of the PCR products with T4 DNA polymerase (Roche,Switzerland), and phosphorylating the DNA termini using polynucleotidekinase (Roche, Switzerland). A pCAGGS expression vector (Niwa H, et al.,Gene. 1991, 108: 193-199) was digested with EcoR I, blunt-ended with aKlenow fragment (Roche, Switzerland), and then dephosphorylated usingbovine small intestine alkaline phosphatase (TAKARA SHUZO, CO., LTD.,Japan). The thus prepared cDNA fragment encoding FGF-23 was ligated to apGAGGS vector. The thus prepared expression vector was cloned and thenucleotide sequence was determined, thereby confirming that a targetsequence encoding the FGF-23 protein was precisely inserted therein.This vector is referred to as pGAGGS/FGF-23.

The above fragment encoding FGF-23 amplified using the FlEcoRI primerand the LNot primer was digested with EcoR I and Not I, and thenpurified. The purified product was cloned by insertion thereof into EcoRI and Not I restriction enzyme sites of a pEAK8/IRES/EGFP vector thathad been prepared by ligating an intramolecular ribosome entry sequence(IRES) and an enhanced-type green fluorescence protein (EGFP) to a pEAK8expression vector (Edge Biosystem, U.S.A.). The nucleotide sequence ofthe obtained plasmid was determined, confirming that it encoded theFGF-23 protein. This vector is referred to as pEAK8/IRES/EGFP/FGF-23.LNot: ATAAGAATGCGGCCGCTCAGATGAACTTGGCGAA (SEQ ID NO: 4)

pGAGGS/FGF-23 was linearized by digestion with EcoR I, and thenblunt-ended using a Klenow fragment (Roche, Switzerland). This wasfurther digested with BamH I. A DNA fragment containing the cDNA ofFGF-23 was separated and purified by agarose electrophoresis.Furthermore, an INPEP4 expression vector was digested with Bgl II,blunt-ended using a Klenow fragment (Roche, Switzerland), digested withBamH I, and then subjected to agarose electrophoresis, thereby purifyingthe vector. The fragment containing FGF-23 cDNA and the vector wereligated. The thus prepared expression vector was cloned, and then thenucleotide sequence was determined, thereby confirming that the targetsequence encoding the FGF-23 protein was precisely inserted therein.This vector is referred to as INPEP4/FGF-23.

(3) Construction of Expression Vector of FGF-23RQH

It has been found that the FGF-23 protein can be easily cleaved betweenthe 179^(th) Arg residue and the 180^(th) Ser residue. The N-terminalside amino acid sequence of the cleavage site isArg176-His177-Thr178-Arg179 (SEQ ID NO: 31), agreeing with theArg-X-X-Arg sequence, which is the recognition sequence of aprotein-converting enzyme. Moreover, it is known that a missensemutation in ADHR is a substitution mutation of the 176^(th) or the178^(th) Arg residue. Hence, we constructed a vector for experimentallypreparing a mutant FGF-23 protein (hereinafter referred to as FGF-23RQH)as a model of the mutant FGF-23 recognized in ADHR, showing resistanceagainst cleavage by a protein-converting enzyme, having His6-tag on theC-terminus, and having Gln residues as a result of substitution of176^(th) and 179^(th) Arg residues. In this preparation method, an RQFforward primer (SEQ ID NO: 5) and an RQR reverse primer (SEQ ID NO: 6)containing nucleotide substitution sequences to be used for substitutingArg with Gln were synthesized. Furthermore, in combination with thesenucleotide substitution primers, ME1 (SEQ ID NO: 7) and HNt (SEQ ID NO:8) primers for amplifying the FGF-23 sequences on the 5′ side and on the3′ side of the mutation introduction site were prepared. ME1 is aforward primer of a portion containing the initiation codon encoded byFGF-23 cDNA, and has an EcoR I restriction enzyme sequence. HNt is areverse primer capable of inserting a codon sequence encoding anHis6-tag sequence before the termination codon encoded by FGF-23 cDNA,and of adding a Not I restriction enzyme sequence. RQF:ATACCACGGCAGCACACCCAGAGCGCCGAG (SEQ ID NO: 5) RQR:CTCGGCGCTCTGGGTGTGCTGCCGTGGTAT (SEQ ID NO: 6) ME1:ATGAATTCCACCATGTTGGGGGCCCGCCTCAGG (SEQ ID NO: 7) HNt:ATGCGGCCGCCTAATGATGATGATGATGATGGATGA (SEQ ID NO: 8) ACTTGGCGAAGGG

PCR reaction was performed using 10 ng of pGAGGS/FGF-23 as a template, acombination of RQF and HNt primers, and a combination of ME1 and RQRprimers (200 nM each). pfu DNA polymerase (Promega, U.S.A.) was used forreaction. After keeping the temperature at 94° C. for 1 minute, 25cycles of a reaction process, each consisting of 94° C. for 30 seconds,55° C. at for 30 seconds, and 72° C. for 1 minute, were conducted. Thethus obtained 2 types of reaction solution were diluted 10 times. Thereaction solutions (1 μl each) were admixed, so as to prepare atemplate. ME1 and HNt were added to a final concentration of 200 nM tothe template, thereby preparing 50 μl of a PCR reaction solution. Afterthe solution was kept at 94° C. for 1 minute, 25 cycles of a PCRreaction process, each cycle consisting of 94° C. for 30 seconds, 55° C.for 30 seconds, and 72° C. for 1 minute, were conducted. Here, LA TaqDNA polymerase (TAKARA SHUZO, CO., LTD., Japan) was used. The thusobtained amplification product of approximately 800 bp was digested withEcoR I and Not I, and then purified, thereby obtaining an insert DNA.This was cloned by insertion thereof into the EcoR I and Not Irestriction enzyme sites of the pEAK8/IRES/EGFP vector that had beenprepared by ligating an intramolecular ribosomal entry sequence (IRES)and an enhanced-type green fluorescence protein (EGFP) to a pEAK8expression vector (Edge Biosystem, U.S.A.). The nucleotide sequence ofthe obtained plasmid was determined, confirming that the 176^(th) and179^(th) Arg had been converted to Gln as expected, and that theyencoded the mutant FGF-23 protein having the His6-tag sequence added tothe C-terminus. This vector is referred to as pEAK8/IRES/EGFP/FGF-23RQH.

EXAMPLE 2 Expression of Recombinant FGF-23 Protein and RecombinantMutant FGF-23 Protein

(1) Obtainment of FGF-23H-Expressing Cells

Approximately 20 μg of pcDNAFGF-23H was linearized by cleaving at an FspI restriction enzyme site within the ampicillin-resistance gene in thevector, and then purified. The purified product was dissolved in 10 μlof pure water, admixed with 1×10⁷ CHO Ras clone-1 cells (Shirahata S.,et al., Biosci Biotech Biochem, 59: 345-347, 1995), and then the genewas introduced into a cell by an electroporation method using GenePulser II (Bio Rad, U.S.A.). After these cells were cultured in a MEMαculture solution (Gibco BRL, U.S.A.) containing 10% FCS for 24 hours,Zeocin (Invitrogen, U.S.A.) was added to a final concentration of 0.5mg/ml to the solution, and the resultant was cultured for 1 week. Cellsthat had adhered and grown were freed using trypsin, and then cloned bya limiting dilution method in the presence of Zeocin at a finalconcentration of 0.3 mg/ml, thereby obtaining 35 types of cloned cells.Cells expressing the FGF-23H protein at the highest levels among thecells were identified by Western blotting shown below. The culturesupernatants of each type of cloned cell were collected, and thensubjected to SDS-polyacrylamide electrophoresis. Then the protein wastransferred to a PVDF membrane (Millipore, U.S.A.), and the signalsderived from the FGF-23H protein at around approximately 32 kDa wasdetected using anti-His-tag (C-terminus) antibodies (Invitrogen, U.S.A.)and an ECL luminescence system (Amersham Pharmacia Biotech, U.S.A.). Asa result, clones referred to as #20 for which the highest expressionlevel had been observed were named CHO-OST311, and then deposited at theInternational Patent Organism Depositary of the National Institute ofAdvanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan) as of Aug. 11, 2000, under theaccession number of FERM BP-7273. In this specification, CHO-OST311 isreferred to as CHO-FGF23H.

(2) Obtainment of Cells Expressing FGF-23 and Cells Expressing FGF-23RQH

pEAK8/IRES/EGFP/FGF-23 and pEAK8/IRES/EGFP/FGF-23RQH vectors wereintroduced into CHO Ras clone-1 cells by a gene transfer method usingmembrane fusion lipids. CHO Ras clone-1 cells were cultured to such anextent that the cells covered approximately 60% of the bottom surfacesof a 6-well plate. Then, the culture solution was removed, and then 1 mlof a serum-free MEMα culture solution was added. 2.5 μg of vectors to beintroduced and 10 μl of Transfectam (trademark) (Promega, U.S.A.) wereseparately admixed with 50 μl of serum-free MEMα culture solutions. Thetwo were then mixed, incubated for 10 minutes, mixed, and then added toa previously prepared 6-well plate. After 2 hours of culture, theculture solutions containing DNA were removed by substitution withculture solutions containing 10% FCS, and then culture was performedovernight. On the next day, puromycin (Sigma, U.S.A.) was added to afinal concentration of 5 μg/ml, thereby selecting drug-resistant cells.The drug-resistant cells obtained in this manner were cloned by alimiting dilution method similarly to the above obtainment of theFGF-23H-expressing cells. Furthermore, cell lines expressing targetproteins at the highest levels were selected by Western blotting. Thesecells are referred to as CHO-FGF23 and CHO-FGF23RQ, respectively.

(3) Purification of Recombinant Protein

When recombinants in the culture supernatants of CHO-FGF23H weredetected by Western blotting using antibodies against the C-terminalHis6-tag sequence, a band at around 32 kDa and a band at around 10 kDawere recognized, as shown in FIG. 1A. When the two bands were excisedfrom gel and the amino acid sequences on the N terminal side weredetermined, an amino acid sequence beginning from the 25^(th) amino acidresidue of SEQ ID NO: 1 was detected in the band of a larger molecularweight, suggesting that the signal sequence had been removed during thesecretion process from the FGF-23 protein. On the other hand, an aminoacid sequence beginning from the 180^(th) amino acid residue in SEQ IDNO: 1 was confirmed in the band of a smaller molecular weight, revealingthat the fragments had been generated by the cleavage between positions179 and 180. By detection using polyclonal antibodies recognizing theN-terminal side of FGF-23, the presence of a peptide thought to have asequence to position 179 was also recognized.

1000 ml of the culture supernatant of the CHO-FGF23H cells was subjectedto centrifugation at 16,200 g for 15 minutes at 4° C., so as to removesuspended cells. The supernatant was then passed through a column(internal diameter of 30 mm×length of 200 mm) filled with SP-sepharoseFF (trademark) (Amersham Pharmacia Biotech, U.S.A.), so that peptidescorresponding to the 180^(th) to the 251^(st) amino acid residues in SEQID NO: 1 and having His6-tag sequence added thereto passed through thecolumn without being adsorbed, while peptides corresponding to the25^(th) to the 251^(st) amino acid residues in SEQ ID NO: 1(hereinafter, may also be referred to as the full-length FGF-23 protein)having an His6-tag added thereto were adsorbed to the column. When theadsorbed substances in the column were eluted with an NaCl concentrationgradient ranging from 0 to 0.7 M in a 50 mM sodium phosphate buffer (pH6.7), the full-length FGF-23 protein having an His6-tag added theretowas observed in fractions eluted with approximately 0.3 M NaCl, peptidesthought to have sequences ranging from the 179^(th) amino acid residueto the N-terminal side of SEQ ID NO: 1 were confirmed in fractionseluted with approximately 0.4 M NaCl. The fractions separated with theSP-Sepharose column in this manner could then be further separated byapplying them to a Talon Superflow (trademark) (Clontech, U.S.A.) metalaffinity column. The sequence ranging from the 179^(th) amino acidresidue to the N-terminal side also had affinity for the metal column,so that it was effective for purification. Further purification wasperformed using an SP-Sepharose column, so that the full-length FGF-23Hcould be obtained as a single band by CBB staining. The results areshown in FIG. 1B.

The FGF-23 protein can also be purified by a similar method. The culturesupernatant of CHO-FGF23 was filtered through a SuperCap (trademark)(Pall Gelman Laboratory, U.S.A.) membrane with a pore size of 0.2 μm,and then the filtered solution was applied to an SP-Sepharose FF(Amersham Pharmacia Biotech, U.S.A.). Substances having weak affinityfor the column were washed with a 50 mM sodium phosphate buffer (pH 6.7)for elution. When protein retained by the column was eluted with an NaClconcentration gradient ranging from 0 to 0.7 M, the full-length FGF-23protein was observed in fractions eluted with approximately 0.3 M NaCl.The protein was adsorbed to a Talon Superflow (trademark) (Clontech,U.S.A.) metal affinity column, washed with a 50 mM sodium phosphatebuffer (pH 6.7), and then varied concentrations of imidazole were addedto the column, thus eluting and purifying the protein. Furthermore,fractions containing target protein were adsorbed to an SP Sepharose FFcolumn, eluted, and then purified. By a similar method, the full-lengthFGF-23RQ protein was purified from the CHO-FGF23RQ supernatant.

EXAMPLE 3 Obtainment of Hybridomas Producing Human Monoclonal AntibodiesAgainst Human FGF-23

Monoclonal antibodies were prepared in this example according to ageneral method as described in Introduction to Monoclonal AntibodyExperimental Protocols (Tamie Ando, et al., “Tan-kurohn Koutai JikkenSosa Nyumon,” KODANSHA, 1991) and the like. Balb/c mice were used asanimals to be immunized. Immunization with human FGF-23 was performed bythe following 2 types of methods depending on differences in immunogens.

(1) Immunization with a Combination of Administration of Vectors andAdministration of Recombinant Protein

Initial immunization was carried out for Balb/c mice by introducing theINPEP4/FGF-23 vectors prepared in Example 1 (10 or 50 μg/mouse)intravenously using Trans IT (trademark) In Vivo Gene Delivery Systemreagent (TAKARA SHUZO, Japan). Booster immunization was performed byintroducing the same vectors once in week 1 after the initialimmunization. Furthermore, the FGF-23 RQH protein (20 to 30 μg/mouse)prepared in Example 2 was suspended in RIBI adjuvants (Corixa, U.S.A.)containing squalene, Tween80, Monophosphoryl lipid A, and Trehalosedimycolate, so as to prepare emulsions. Booster immunization wasperformed 4 or 5 times by intraperitoneal injection of the emulsions.Subsequently, on day 4 before the obtainment of splenocytes describedbelow, mice were immunized by tail intravenous injection of the FGF-23Hprotein (18 μg/mouse) prepared in Example 2.

(2) Immunization using Human FGF-23

Initial immunization was performed for Balb/c mice by intraperitonealinjection of a suspension prepared by suspending FGF-23 (22 μg/mouse)prepared in Example 2 in the above RIBI adjuvants. Furthermore, boosterimmunization was performed once every week by intraperitoneal injectionof the same protein over a period of 4 weeks. On day 3 before theobtainment of splenocytes as described below, immunization was performedby tail intravenous injection of FGF-23 (10 μg/mouse).

(3) Preparation and Selection of Hybridoma

Spleens were excised from the mice immunized as described above.Splenocytes collected from the spleens were mixed at a 5:1 proportion tomouse myeloma SP2/0 (ATCC: CRL 1581), and then the cells were fusedusing polyethylene glycol 1500 (Roche Diagnostics, Japan) as a fusionagent, thereby preparing hybridomas. The hybridomas were selected byculturing the cells in HAT-containing DMEM media (Gibco BRL, U.S.A.)containing 10% Fetal Calf Serum (FCS), hypoxanthine (H), aminopterin(A), and thymidine (T). Furthermore, cloning was performed by a limitingdilution method using HT-containing DMEM media. Thus, cloned hybridomasderived from a single cell were obtained.

(4) Selection of Cloned Hybridoma Producing Anti-FGF-23 Antibodies

Hybridomas producing antibodies specifically recognizing the FGF-23protein was selected by examining the binding between antibodiesproduced by the hybridomas and the FGF-23 protein. Selection ofhybridomas obtained by immunization conducted according to the 1 stmethod above was conducted as follows. 50 μl of a solution of theFGF-23H protein diluted to a concentration of 1 μg/ml in a 50 mM NaHCO3solution was added to each well of a 96-well microplate for ELISA(Maxisorp (trademark), Nunc, U.S.A.). Incubation was performed at 37° C.for 30 minutes or 4° C. for 12 hours, so that the FGF-23H protein wasadsorbed to the microplate. Next, the solution was removed, a blockingreagent (SuperBlock (trademark) Blocking Buffer, PIERCE, U.S.A.) wasadded to each well, and then incubation was performed at roomtemperature for 30 minutes. Each well was then washed twice withTris-buffered saline containing 0.1% Tween20 (500 mM NaCl-containingTRIZMA pre-set crystals (trademark), Sigma, U.S.A.) (T-TBS). 50 μl ofthe culture supernatant of each type of hybridoma was added to each wellof the microplate that had been coated with the FGF-23H protein asdescribed above. After 30 minutes of reaction, each well was washedtwice with T-TBS. Subsequently, 50 μl of peroxidase-labeled goatanti-mouse IgG antibodies (Zymed laboratories, U.S.A.) diluted3,000-fold was added to each well, followed by incubation at roomtemperature for 30 minutes. The wells were washed 3 times with T-TBS and50 μl of a substrate buffer containing tetramethylbenzidine (Denmark,DAKO) was added to each well, followed by incubation at room temperaturefor 15 minutes. Next, 50 μl of 0.5 M sulfuric acid was added to eachwell, so as to stop reaction. Absorbance at a wavelength of 450 nm wasmeasured using a microplate reader (MTP-300, CORONA ELECTRIC CO., LTD.,Japan) with a reference wavelength of 570 nm. Here, the hybridomasshowing clear increases in absorbance were selected, and a similarexperiment was conducted using the FGF-23 protein, so that clones forwhich binding with FGF-23 had been re-confirmed were selected. Thus, 9types of clone were obtained as hybridomas producing antibodiesrecognizing the FGF-23 protein.

Among these clones, 1C3H, 1D6A, 2A2B, 2C3B, and 2C5L described belowwere included.

Hybridomas obtained by immunization conducted according to the 2ndmethod above were selected as follows. 50 μl of a solution of the FGF-23protein diluted to a concentration of 1 μg/ml in a 50 mM NaHCO3 solutionwas added to each well of a 96-well microplate for ELISA (Maxisorp(trademark), Nunc, U.S.A.). Incubation was performed at 4° C. for 10hours, so that the FGF-23 protein was adsorbed to the microplate. Next,the solution was removed, a blocking reagent (SuperBlock (trademark)Blocking Buffer, PIERCE, U.S.A.) was added to each well, and thenincubation was performed at room temperature for 30 minutes. Each wellwas then washed twice with Tris-buffered saline (T-TBS) containing 0.1%Tween20. 50 μl of the culture supernatant of each type of hybridoma wasadded to each well of the microplate that had been coated wit theFGF-23H protein. After 30 minutes of reaction, each well was washedtwice with Tris-buffered saline (T-TBS) containing 0.1% Tween20.Subsequently, 50 μl of peroxidase-labeled goat anti-mouse IgG antibodies(Zymed laboratories, U.S.A.) diluted 3,000-fold was added to each well,followed by incubation at room temperature for 30 minutes. The wellswere washed 3 times with T-TBS and 50 μl of a substrate buffercontaining tetramethylbenzidine (Denmark, DAKO) was added to each well,followed by incubation at room temperature for 15 minutes. Next, 50 μlof 0.5 M sulfuric acid was added to each well, so as to stop reaction.Absorbance at a wavelength of 450 nm was measured using a microplatereader (MTP-300, CORONA ELECTRIC CO., LTD., Japan) with a referencewavelength of 570 nm. Here, hybridomas showing clear increases inabsorbance were selected. Thus, 4 types of new clones were obtained ashybridomas producing antibodies recognizing the FGF-23 protein. Amongthese clones, 3C1E was included.

The subclasses of the thus obtained antibodies that specificallyrecognize the FGF-23 protein was identified using an Iso Strip mousemonoclonal antibody isotyping kit (Roche, U.S.A.). The results are shownin Table 1. TABLE 1 Anti-human FGF-23 antibodies Hybridoma cloneSubclass ELISA 450 nm-570 nm 1C3H IgG1(κ) 3.39 1D6A IgG1(κ) 3.21 2A2BIgG1(κ) 2.67 2C3B IgG1(κ) 1.21 3C1E IgG1(κ) 3.5 or more 2C5L IgG1(κ)1.38

Among the above hybridoma clones, 3 hybridoma clones (2C3B, 3C1E, and1D6A) were internationally deposited under the Budapest Treaty at theInternational Patent Organism Depositary of the National Institute ofAdvanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan) as of Dec. 26, 2001. Furthermore, the2C5L hybridoma clone was deposited internationally under the BudapestTreaty at the International Patent Organism Depositary of the NationalInstitute of Advanced Industrial Science and Technology (Tsukuba Central6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) as of Jan. 6, 2003. Theaccession numbers of these clones are as follows.

-   -   2C3B: FERM BP-7838    -   3C1E: FERM BP-7839    -   1D6A: FERM BP-7840    -   2C5L: FERM BP-8268

EXAMPLE 4 Preparation of Monoclonal Antibodies

(1) Preparation of Culture Supernatants Containing Anti-FGF-23Antibodies

Hybridomas producing anti-FGF-23 antibodies were acclimatized in eRDFmedia (KYOKUTO PHARMACEUTICAL INDUSTRIAL CO., LTD. Japan) containing 10μg/ml bovine insulin (Sigma, U.S.A.), 5.5 μg/ml human transferrin(Sigma, U.S.A.), 0.01 mM ethanolamine (Sigma, U.S.A.), and 5 ng/mlsodium selenite (Sigma, U.S.A.). Hybridomas for the preparation ofantibodies were cultured in spinner flasks. The culture solution waspassed through a filter with a pore size of 0.2 μm (Pall GelmanLaboratory, U.S.A.) so as to remove waste matters such as hybridomas,thereby collecting culture supernatants containing antibodies.

(2) Purification of Monoclonal Antibodies using Protein G

The culture supernatant containing anti-FGF-23 antibodies was passedthrough a protein G Sepharose 4FF column (Amersham Pharmacia Biotech,U.S.A.), so that the antibodies were adsorbed to the column. Theantibodies were then eluted using a 0.1 M glycine buffer (pH 2.8). 1 MTris-HCl was added to the elution fractions to adjust the pH to 7.2. Thethus prepared antibody solution was dialyzed and substituted with PBS(−)using a dialysis membrane with a molecular weight cutoff of 10000(Spectrum Laboratories, U.S.A.) and filter sterilized using a MILLEX-GVmembrane filter with a pore size of 0.22 μm (Millipore, U.S.A.), therebyobtaining purified anti-FGF-23 antibodies. The concentrations ofpurified antibodies were calculated by measuring absorbance at 280 nm,followed by calculation with 1 mg/ml as 1.35 OD.

(3) Purification of Monoclonal Antibodies Using Protein A

Antibodies were affinity-purified from the culture supernatantcontaining anti-FGF-23 antibodies using a protein A carrier column (IBLCo., Ltd., Japan), a glycine buffer (pH 8.9) as adsorption buffer, and acitric acid buffer (pH 4.0) as an elution buffer. 1 M Tris-HCl was addedto the elution fraction containing the antibodies, so as to adjust pH toaround 7.2. Then, the solution containing the antibodies was substitutedwith PBS(−) using a dialysis membrane and filter-sterilized with amembrane filter with a pore size of 0.22 μm, thereby obtaining purifiedanti-FGF-23 antibodies. The concentrations of the purified antibodieswere calculated by measuring absorbance at 280 nm, followed bycalculation with 1 mg/ml as 1.35 OD.

Each of the thus obtained purified monoclonal antibodies is expressedusing the name of the hybridoma producing the given antibody. Forexample, antibodies produced by 1 D6A hybridomas are described as 1D6Aantibodies.

EXAMPLE 5 Preparation of Anti-FGF-23 Partial Peptide PolyclonalAntibodies

(1) Synthesis of Peptides Corresponding to FGF-23 Partial Sequence

The degree of hydrophobicity of the polypeptide of SEQ ID NO: 1 waspredicted using the calculator function of MacVector version 6.5.1, andthen sites appropriate for the preparation of peptide antibodies werepredicted. With the condition that sites that have high degree ofhydrophilicity and can be subjected to sugar chain modification orphosphorylation are excluded, partial sequences inferred to beappropriate for the preparation of antibodies were extracted. As aresult, an hFGF23-25 peptide (SEQ ID NO: 9) comprising 15 amino acidresidues beginning from the 25^(th) tyrosine (residue number: 25) of SEQID NO: 1 and having a cysteine residue added to the C-terminus of thepeptide, an hFGF23-48 peptide (SEQ ID NO: 10) comprising 20 amino acidresidues beginning from the 48^(th) arginine and having a cysteineresidue added to the C-terminus of the peptide, an hFGF23-114 peptide(SEQ ID NO: 11) comprising 15 amino acid residues beginning from the114^(th) arginine and having a cysteine residue added to the C-terminusof the peptide, an hFGF23-148 peptide (SEQ ID NO: 12) comprising 16amino acid residues beginning from the 148^(th) glycin and having acysteine residue added to the C-terminus of the peptide, an hFGF23-170peptide (SEQ ID NO: 13) comprising 10 amino acid residues beginning fromthe 170^(th) asparagines and having a cysteine residue added to theN-terminus of the peptide, an hFGF23-174 peptide (SEQ ID NO: 14)comprising 14 amino acid residues beginning from the 174^(th) prolineand having a cysteine residue added to the C-terminus of the peptide, anhFGF23-180 peptide (SEQ ID NO: 15) comprising 15 amino acid residuesbeginning from the 180^(th) serine and having a cysteine residue addedto the C-terminus of the peptide, an hFGF23-210 peptide (SEQ ID NO: 16)comprising 13 amino acid residues beginning from the 210^(th) Leu andhaving a cysteine residue added to the C-terminus of the peptide, and anhFGF23-237 peptide (SEQ ID NO: 17) comprising 15 amino acid residuesbeginning from the 237^(th) glycine were selected as antigens, andchemically synthesized. hFGF23-25: YPNASPLLGSSWGGLC (SEQ ID NO: 9)hFGF23-48: RNSYHLQIHKNGHVDGAPHQC (SEQ ID NO: 10) hFGF23-114:RFQHQTLENGYDVYHSPQYHC (SEQ ID NO: 11) hFGF23-148: GMNPPPYSQFLSRRNEC (SEQID NO: 12) hFGF23-170: CNTPIPRRHTR (SEQ ID NO: 13) hFGF23-174:PRRHTRSAEDDSERC (SEQ ID NO: 14) hFGF23-180: SAEDDSERDPLNVLKC (SEQ ID NO:15) hFGF23-210: LPSAEDNSPMASDC (SEQ ID NO: 16) hFGF23-237:GGTGPEGCRPFAKFI (SEQ ID NO: 17)(2) Preparation of Polyclonal Antibodies Against Anti-FGF-23 PartialPeptide

All the above peptides were bound to bovine thyroglobulin, the carrierprotein, via their own cysteine residues, and then used forimmunization. For immunization, 3 rabbits were used per antigen peptide.Initial immunization was conducted by preparing emulsions (100μg/rabbit) using peptides bound to the carrier protein using Freund'scomplete adjuvants, and intradermally or subcutaneously administeringthe emulsions to rabbits. 1 week after the initial immunization,emulsions prepared using 100 μg of peptides bound to the carrier proteinusing Freund's incomplete adjuvants were similarly administered. Thesame administration was conducted 6 times at intervals of 2 weeks, andthen exsanguination was performed 1 week after the final administration,thereby preparing anti-serum.

To prepare an affinity column for the purification of anti-FGF-23partial peptide antibodies from rabbit serum, each peptide used forimmunization was immobilized on gel using a SulfoLink Kit (PIERCE,U.S.A.). Anti-serum was added to the column using PBS(−) as anadsorption buffer, so as to retain antibodies binding to the peptidesused for immunization in the column. Next, the antibodies bound to thecolumn were eluted using a 0.1 M glycine buffer (pH 2.5 to 3.3) as anelution buffer, and were then collected. 1 M Tris-HCl was added to theeluted fractions to adjust pH to around 7.2. The thus prepared antibodysolution was subjected to a NAP25 gel filtration column (AmershamPharmacia Biotech, U.S.A.), so that the buffer was substituted withPBS(-). Filter sterilization was performed using a MILLEX-GV membranefilter (Millipore, U.S.A.) with a pore size of 0.22 μm, therebyobtaining antibodies against each peptide. The concentrations ofpurified antibodies were calculated by measuring absorbance at 280 nm,followed by calculation with 1 mg/ml as 1.35 OD. Each of the thusobtained purified antibodies is expressed using the name of a peptideused for immunization. For example, an antibody obtained by theimmunization with the hFGF23-25 peptide of SEQ ID NO: 9 is described asan hFGF23-25 antibody.

(3) Recognition of FGF-23 Protein and Metabolites thereof by anti-FGF-23Partial Peptide Antibodies

The FGF-23H protein and metabolites thereof in the supernatant ofCHO-FGF23H-expressing cells were analyzed by Western blotting using thehFGF23-48 and hFGF23-148 antibodies obtained by the above method. Asshown in FIG. 1A, full-length FGF-23H protein of around 32 kDa wasdetected with both of these antibodies. Furthermore, metabolites with asize of around 18 kDa or less were observed, and they were considered tobe derived from fragments on the N-terminal side resulting from thecleavage of the FGF-23 protein between the 179^(th) and the 180^(th)amino acid residues of the amino acid sequence of SEQ ID NO: 1. ThehFGF23-48 antibody recognized smaller fragmented metabolites. FGF-23RQprotein and metabolites thereof in the culture supernatant ofCHO-FGF23RQ producing mutant protein avoiding cleavage between the179^(th) and 180^(th) amino acid residues were examined using hFGF23-148antibodies. As shown in FIG. 1C, fragmented peptides that had beenrecognized in the case of FGF-23H became undetected. Based on the aboveresult, it is considered that in the metabolism by the cleavage of theFGF-23 protein, the N-terminal fragments generated by cleavage betweenthe 179^(th) and 180^(th) amino acid residues are further fragmentedinto smaller sizes, but such small fragments are generated aftercleavage has occurred between the 179^(th) and the 180^(th) amino acidresidues. Hence, it was revealed that determining the presence or theabsence of cleavage between the 179^(th) and the 180^(th) amino acidresidues is very important when considering the metabolism of the FGF-23protein.

EXAMPLE 6 Preparation of Biotinylated Antibodies

Biotinylation was performed using the 9 above-purified types ofpolyclonal antibodies against the FGF-23 partial peptides and some of 13types of anti-FGF-23 monoclonal antibodies. 10 μl of a solution that hadbeen prepared by dissolving Biotin-AC5-Osu (DOJINDO LABORATORIES. Japan)to dimethylformamide at a concentration of 1.82 mg/ml was added to 1 mlof an antibody solution that had been diluted to a concentration of 1mg/ml with a 50 mM sodium hydrogen carbonate solution, and then theresultants were admixed by being turned upside down at 4° C. for 2hours. Subsequently, the reaction solution was applied to a NAP10 column(Amersham Pharmacia Biotech, U.S.A.), unreacted Biotin-AC5-Osu wasremoved, and the solvent was substituted with PBS(−). Thus, 9 types ofbiotin-labeled anti-FGF-23 partial peptide polyclonal antibodies and 5types of biotin-labeled anti-FGF-23 monoclonal antibodies (1C3Hantibody, 1D6A antibody, 2A2B antibody, 2C3B antibody, and 3C1Eantibody) were obtained.

EXAMPLE 7 Sandwich ELISA Method Using Polyclonal Antibodies RecognizingSpecific Site of FGF-23

(1) Construction of Sandwich ELISA System

The construction of a sandwich ELISA system was examined by combining 8types of antibodies (hFGF23-25 antibody, hFGF23-48 antibody, hFGF23-114antibody, hFGF23-148 antibody, hFGF23-170 antibody, hFGF23-180 antibody,hFGF23-210 antibody, and hFGF23-237 antibody) as antibodies forimmobilization and antibodies for detection, among the above 9 types ofpolyclonal antibodies against the FGF-23 partial peptide sequences.

To immobilize antibodies, 8 types of anti-FGF-23 partial peptidepolyclonal antibodies (hFGF23-25 antibody, hFGF23-48 antibody,hFGF23-114 antibody, hFGF23-148 antibody, hFGF23-170 antibody,hFGF23-180 antibody, hFGF23-210 antibody, and hFGF23-237 antibody) werediluted to 30 μg/ml with a 50 mM sodium hydrogen carbonate solution. 50μl of the solution was added per well of a 96-well plate for ELISA(Maxisorp (trademark), Nunc, U.S.A.), and then incubated at 37° C. for1.5 hours. Subsequently, the reaction solution was removed, 50 μl ofSuperBlock (trademark) (PIERCE, U.S.A.) was added per well, 60 minutesof incubation was performed at room temperature, so that blocking wasconducted. After the solution was removed, 50 μl of 1 μg/ml FGF-23Hprotein was added per well and incubated at room temperature for 1 hour,so as to bind the protein with the immobilized antibodies. Afterantibody reaction, the wells were washed 3 times with T-TBS. The above 8types of biotin-labeled anti-FGF-23 partial peptide antibodies(hFGF23-25 antibody, hFGF23-48 antibody, hFGF23-114 antibody, hFGF23-148antibody, hFGF23-170 antibody, hFGF23-180 antibody, hFGF23-210 antibody,and hFGF23-237 antibody) were diluted with T-TBS containing 10% Blockace(DAINIPPON PHARMACEUTICAL CO., LTD. Japan) to 10 μg/ml. These antibodiesand a T-TBS solution containing 10% Blockace (DAINIPPON PHARMACEUTICALCO., LTD. Japan) as a control were separately added at 50 μl per well.Each solution was incubated at room temperature for 30 minutes, so as toconduct secondary antibody reaction. After each well was washed 3 timeswith T-TBS, 50 μl of HRP-labeled streptavidin (DAKO, Denmark) diluted5000-fold with T-TBS containing 10% Blockace was added per well. Thesolutions were incubated at room temperature for 30 minutes, so as tobind the streptavidin with biotin-labeled antibodies. Each well waswashed 4 times with T-TBS and 50 μl of tetramethylbenzidine (DAKO,Denmark), which is a peroxidase chromogenic substrate, was added perwell so as to cause color development at room temperature for 15minutes. 50 μl of 0.5 M sulfuric acid solution was added per well, so asto stop reaction. Measurement was conducted using MTP-300 (system formeasuring absorbance) for a 96-well plate (CORONA ELECTRIC CO., LTD.,Japan), and values were obtained by subtracting absorbance at 570 nmfrom absorbance at 450 nm (Table 2). In the case of the control to whichbiotin-labeled antibodies had not been added, all the values obtainedfor 450 nm-570 nm were 0.06 or less. However, as shown in Table 2, inthe case of multiple combinations of immobilized antibodies andantibodies for detection, values obtained for 450 nm-570 nm weresignificantly elevated. As shown in FIG. 1, since polypeptides generatedby the cleavage of the FGF-23 protein are known to be present,polypeptides of different molecular types derived from FGF-23 may bepresent in the same sample. Based on the fact that the antibodies usedherein recognize specific sites of FGF-23, molecular types to bemeasured can be narrowed according to combinations of the antibodies.For example, when a combination is employed, where hFGF23-170 antibodyare immobilized and detection is performed using hFGF23-25 antibody,since the antigen sites of both antibodies are contained in theN-terminal side partial polypeptide fragments between the 25^(th) andthe 179^(th) residues of the amino acid sequence of the FGF-23 proteinrepresented by SEQ ID NO: 1, it is predicted that not only thefull-length polypeptide between the 25^(th) and the 251^(st) residues ofSEQ ID NO: 1, but also the N-terminal partial polypeptide fragments, canbe detected by the sandwich ELISA using this combination. On the otherhand, when hFGF23-180 antibody is immobilized, and detection isperformed using hFGF23-237 antibody, it is predicted that not onlyfull-length polypeptides, but also a C-terminal partial polypeptidefragment corresponding to the 180^(th) to the 251^(st) residues of theFGF-23 protein represented by SEQ ID NO: 1, can be detected. Moreover,for example, when hFGF23-237 antibody is immobilized and detection isperformed using hFGF23-25 antibody, it is predicted that only thefull-length FGF-23 protein can be detected without detecting N- andC-terminal side partial polypeptides generated after cleavage.Therefore, the composite use of these combinations enables measurementof the absolute quantities of FGF-23 full-length polypeptides andpartial polypeptides in analytes such as biological samples, and alsoenables the ability to distinguish existence ratios of thesepolypeptides. TABLE 2 Detection of FGF-23 protein by sandwich ELISAusing combinations of anti-FGF-23 polyclonal antibodies (A450 nm-A570nm) Biotinylated Immobilized antibodies antibodies hFGF23-25 hFGF23-48hFGF23-114 hFGF23-148 hFGF23-170 hFGF23-180 hFGF23-210 hFGF23-237 NonehFGF23-25 0.517 0.046 0.040 0.050 1.543 1.938 0.686 1.808 0.048hFGF23-48 0.037 0.050 0.028 0.028 0.030 0.033 0.031 0.034 0.037hFGF23-114 0.033 0.026 0.029 0.026 0.027 0.029 0.026 0.029 0.036hFGF23-148 0.091 0.070 0.046 0.140 0.157 0.102 0.083 0.104 0.047hFGF23-170 0.444 0.035 0.033 0.068 0.112 0.054 0.041 0.057 0.042hFGF23-180 0.370 0.036 0.034 0.042 0.045 0.193 0.286 0.652 0.038hFGF23-210 0.309 0.034 0.033 0.036 0.043 0.563 0.065 0.383 0.035hFGF23-237 1.096 0.061 0.047 0.081 0.113 2.143 0.407 0.442 0.057

Types of immobilized antibodies are shown in the top horizontal line,and types of antibodies used for detection are shown in the leftmostcolumn. Figures in this table are measured values obtained by the use ofeach combination.

(2) Quantitative Detection of FGF-23 Protein by Sandwich ELISA

According to the above method for preparing the sandwich ELISA system,FGF-23H protein solutions having concentrations of 1, 0.33, 0.1, 0.033,0.01, 0.0033, and 0.001 μg/ml, respectively, were measured as testsubstances by immobilizing hFGF23-25 antibody and using hFGF23-237antibody for detection. The results are shown in FIG. 2. Within therange of 0.1 to 1 μg/ml, concentration-dependent increases were observedin values obtained by 450 nm-570 nm, revealing that at least within thisconcentration range, the FGF-23H protein can be detected in aconcentration-dependent manner.

EXAMPLE 8 Preparation of FGF-23 Partial Peptide

In addition to the peptides corresponding to the FGF-23 partialsequences prepared in Example 5, peptides having the following partialsequences of FGF-23 were chemically synthesized:

An hFGF23-38 peptide (SEQ ID NO: 18) comprising 13 amino acid residuesbeginning from residue number 38 (glycine) of SEQ ID NO: 1 and having acysteine residue added to the C-terminus of the peptide; an hFGF23-68peptide (SEQ ID NO: 19) comprising 28 amino acid residues beginning fromresidue number 68 (threonine) of SEQ ID NO: 1; an hFGF23-96 peptide (SEQID NO: 20) comprising 18 amino acid residues beginning from residuenumber 96 (methionine) of SEQ ID NO: 1; an hFGF23-129 peptide (SEQ IDNO: 21) comprising 22 amino acid residues beginning from residue number129 (serine) of SEQ ID NO: 1 and having a cysteine residue added to theC-terminus of the peptide; an hFGF23-161 peptide (SEQ ID NO: 22)comprising 13 amino acid residues beginning from residue number 161(arginine) of SEQ ID NO: 1 and having a cysteine residue added to theC-terminus of the peptide; an hFGF23-197 peptide (SEQ ID NO: 23)comprising 16 amino acid residues beginning from residue number 197(alanine) of SEQ ID NO: 1; and an hFGF23-220 peptide (SEQ ID NO: 24)comprising 24 amino acid residues beginning from residue number 220(serine) of SEQ ID NO: 1. hFGF23-38: GLIHLYTATARNSC (SEQ ID NO: 18)hFGF23-68: TIYSALMIRSEDAGFVVITGVMSRRYLC (SEQ ID NO: 19) hFGF23-96:MDFRGNIFGSHYFDPENC (SEQ ID NO: 20) hFGF23-129: SPQYHFLVSLGRAKRAFLPGMNC(SEQ ID NO: 21) hFGF23-161: RNEIPLIHFNTPIC (SEQ ID NO: 22) hFGF23-197:ARMTPAPASCSQELPS (SEQ ID NO: 23) hFGF23-220: SDPLGVVRGGRVNTHAGGTGPEGC(SEQ ID NO: 24)

EXAMPLE 9 Determination of FGF-23 Recognition Region of MonoclonalAntibodies

The regions containing amino acid sequences recognized by anti-FGF-23monoclonal antibodies were determined by examining reactivity withpeptides containing the partial sequences of human FGF-23.

(1) Experiment 1: Binding with Immobilized Peptides

Peptides (SEQ ID NOS: 7 to 17) synthesized in Example 5 were diluted toa concentration of 1 μg/ml with a 50 mM sodium hydrogencarbonatesolution. 50 μl of the solution was added per well of a 96-well platefor ELISA (Maxisorp (trademark), Nunc, U.S.A.), and then the solutionswere incubated at 37° C. for 1 hour, so as to immobilize the FGF-23partial peptides. Next, the solutions were removed, 50 μl of a blockingsolution (Superblock (trademark), PIERCE, U.S.A.) was added per well,and then incubation was performed at room temperature for 60 minutes,thereby performing blocking. After the solutions were removed, 50 μl ofthe culture supernatant of the hybridomas obtained in Example 3 or anHT-containing DMEM medium as a control was added per well, and then thesolutions were incubated at room temperature for 1 hour for binding withthe FGF-23 partial peptides. After the end of antibody-binding reaction,the wells were washed three times with T-TBS. Subsequently, 50 μl ofHRP-labeled goat anti-mouse IgG(H+L)-F(ab′)2 diluted 3000-fold withT-TBS containing a 10% blocking solution (Blockace (trademark),(DAINIPPON PHARMACEUTICAL CO., LTD. Japan) was added per well, and thenthe solutions were incubated at room temperature for 30 minutes so as toresult in binding with the secondary antibody. Each well was washed 3times with T-TBS, 50 μl of tetramethylbenzidine (Denmark, DAKO), whichwas a peroxidase chromogenic substrate, was added per well, and this wasfollowed by color development at room temperature for 3 minutes. Next,50 μl of a 0.5 M sulfuric acid solution was added per well, so as tostop reaction. Measurement was conducted using a system for measuringabsorbance for a 96-well plate (MTP-300, CORONA ELECTRIC CO., LTD.,Japan), and values were obtained by subtracting absorbance at 570 nmfrom absorbance at 450 nm. In the case of the control where only theHT-containing DMEM medium had been added, all the values obtained by 450nm-570 nm were 0.06 or less. However, clearly increased absorbances wereobserved in the cases where the culture supernatants of the hybridoamshad been added and specific peptides had been immobilized. The resultsare shown in Table 3. The 2A2B antibody was bound to hFGF23-148 peptideof SEQ ID NO: 12. 1D6A showed binding with an hFGF23-237 peptide of SEQID NO: 17. Hence, it is concluded that an antibody produced by the 2A2Bhybridoma binds to a region between the 148^(th) and the 163^(rd) aminoacid residues of FGF-23 represented by SEQ ID NO: 1 or a part thereof.Moreover, 1C3H binds to a region between the 180^(th) and the 194^(th)amino acid residues of FGF-23 represented by SEQ ID NO: 1 or a partthereof, and 1D6A binds to a region between the 237^(th) and the251^(st) amino acid residues of FGF-23 represented by SEQ ID NO: 1 or apart thereof. TABLE 3 Reactivity of immobilized peptides having partialsequence of FGF-23 with antibody in the culture supernatant ofmonoclonal-antibody-producing hybridoma Culture Immobilized peptidessupernatant hFGF23-25 hFGF23-48 hFGF23-114 hFGF23-148 hFGF23-170hFGF23-180 hFGF23-210 hFGF23-237 1C3H 0.053 0.043 0.043 0.041 0.0370.240 0.039 0.036 1D6A 0.056 0.051 0.049 0.045 0.044 0.040 0.038 2.0582A2B 0.052 0.048 0.043 2.321 0.040 0.037 0.036 0.040 2C3B 0.056 0.0460.043 0.038 0.039 0.038 0.041 0.041 Control 0.059 0.047 0.048 0.0510.043 0.044 0.045 0.043(2) Experiment 2: Binding with Immobilized Peptides

For FGF-23, cleavage is observed between the 179^(th) and the 180^(th)amino acid residues of the sequence represented by SEQ ID NO: 1. For thepurpose of examining in detail antibodies recognizing a region from thecleavage site to the C-terminal side, a binding experiment was conductedusing synthetic peptides having sequences corresponding to parts of theregion. Peptides (SEQ ID NOS: 14, 15, 16 and 17) synthesized in Example5 and peptides (SEQ ID NOS: 23 and 24) synthesized in Example 8 werediluted to a concentration of 1 μg/ml with a 50 mM sodiumhydrogencarbonate solution. 50 μl of the solution was added per well toa 96-well plate for ELISA (Maxisorp (trademark), Nunc, U.S.A.), and thenthe solutions were incubated at 4° C. for 12 hours for immobilizationonto the plate. Next, the solutions were removed, 50 μl of a blockingsolution (Superblock (trademark), PIERCE, U.S.A.) was added per well,and then incubation was performed at room temperature for 60 minutes,thereby performing blocking. After the solution was removed, 50 μl of asolution that had been prepared by diluting the 1D6A and 3C1E antibodiespurified and obtained in Examples 3 and 4 with T-TBS containing 10%blocking solution (Blockace (trademark), (DAINIPPON PHARMACEUTICAL CO.,LTD. Japan) to a concentration of 10 pg/ml was added per well. As acontrol, a well to which 50 μl of T-TBS containing a 10% blockingsolution (Blockace (trademark), (DAINIPPON PHARMACEUTICAL CO., LTD.Japan) had been added was provided. The solutions were incubated at roomtemperature for 1 hour, thereby conducting a reaction of immobilizedpeptides with antibodies. After the end of the antibody reaction, thewells were washed 4 times with T-TBS, 50 μl of HRP-labeled goatanti-mouse IgG(H+L)-F(ab′)2 diluted 3000-fold with T-TBS containing a10% blocking solution (Blockace (trademark), (DAINIPPON PHARMACEUTICALCO., LTD. Japan) was added per well. The solutions were incubated atroom temperature for 60 minutes for reaction with the secondaryantibody. After each well was washed 4 times with T-TBS, 50 μl oftetramethylbenzidine (Denmark, DAKO), which is a peroxidase chromogenicsubstrate, was added per well, followed by color development at roomtemperature for 20 minutes. 50 μl of a 0.5 M sulfuric acid solution wasthen added per well, so as to stop reaction. Measurement was conductedusing a system for measuring absorbance for a 96-well plate (MTP-300,CORONA ELECTRIC CO., LTD., Japan), and values were obtained bysubtracting absorbance at 570 nm from absorbance at 450 nm. The resultsare shown in Table 4. In the case of the wells used as controls whereonly the solution for dilution had been added, all the values obtainedby 450 nm-570 nm were 0.05 or less. In contrast, the 3C1E antibody wasbound with hFGF23-180 peptide (SEQ ID NO: 15) and the 1D6A antibody wasbound with hFGF23-237 peptides (SEQ ID NO: 17). TABLE 4 Reactivity ofimmobilized peptides having partial sequence of FGF-23 with purifiedmonoclonal antibodies Purified Immobilized peptides antibodieshFGF23-170 hFGF23-174 hFGF23-180 hFGF23-197 hFGF23-210 hFGF23-220hFGF23-237 Control 1D6A 0.041 0.051 0.047 0.050 0.055 0.054 3.412 0.0603C1E 0.037 0.043 0.203 0.041 0.040 0.051 0.042 0.045 Control 0.038 0.0410.035 0.036 0.042 0.048 0.043 0.048

EXAMPLE 10 Detection of FGF-23 Protein and Polypeptide Derived therefromby Immunoprecipitation

Immunoprecipitation was performed so that precipitated protein wasdetected by Western blotting in order to reveal the reactivity of theobtained monoclonal antibodies with the FGF-23 protein and polypeptidesderived therefrom in a liquid phase that was closer to physiologicalconditions compared with immobilized peptides.

The CHO-FGF23 cells expressing FGF-23H prepared in Example 2 werecultured in CHO-S-SFM II media (Gibco BRL, U.S.A.) for 4 days, therebyobtaining the culture supernatant containing the FGF-23 protein,N-terminal side polypeptide fragments, and C-terminal side polypeptidefragments. 0.5 μg each of the 5 types of anti-FGF-23 monoclonalantibodies prepared in Examples 3 and 4 was added per 400 μl of thesupernatant, so as to prepare solutions. A control solution was preparedby adding only a buffer. These solutions were admixed by being turnedupside down at 4° C. for 1.5 hours so as to cause the antibodies toreact with the protein in the culture supernatant. Subsequently, 30 μlof protein G Sepharose 4FF resins (Amersham Pharmacia Biotech, U.S.A.)was added, the solutions were admixed by being turned upside down at 4°C. for 3 hours, and then substances not bound to resins were washed off3 times with PBS. To a half quantity of the resins, 30 μl of 20 mMDTT-containing buffer and 20 mM DTT-free sample buffer (50 mM Tris-ClpH6.8, 1% SDS, 10% glycerol, 0.001% bromophenol blue, 2 mM EDTA) wereadded. The solutions were heated at 95° C. for 5 minutes and thencentrifuged, thereby collecting supernatants. The thus collectedimmunoprecipitates were separated by 10% to 20% concentration-gradientpolyacrylamide gel electrophoresis. Protein in the gel were transferredto an Immobilon PVDF membrane (Millipore, U.S.A.) using a Semi DryBlotting System (Owl Separation Systems, U.S.A.). After blocking thePVDF membrane with a blocking solution (Blockace (trademark), (DAINIPPONPHARMACEUTICAL CO., LTD. Japan), incubation was performed at 4° C. for12 hours in a solution of biotin-labeled 2A2B antibody or 1C3Hantibodies that had been diluted to 1 μg/ml in T-TBS. Furthermore,HRP-labeled streptavidin (DAKO, Denmark) was allowed to react with theresultants. After washing, the resultant was exposed to a film using anECL Plus luminescence system (Amersham Pharmacia Biotech, U.S.A.) for 1hour, and then the film was developed using an automatic processor (FUJIPHOTO FILM CO., LTD., Japan). The results are shown in FIG. 3. The 2A2Bantibody used in Western blotting recognizes a site corresponding toamino acid residue numbers 148 to 163 of SEQ ID NO: 1. Moreover, the1C3H antibody used herein was shown to recognize a site corresponding toamino acid residue numbers 180 to 194 of SEQ ID NO: 1. By the use ofboth antibodies, fragmented polypeptides generated by cleavage betweenthe 179^(th) and the 180^(th) amino acid residues can be distinguishedfrom other polypeptides and recognized. As a result of this experiment,monoclonal antibodies were classified into the following 3 types.Specifically, these are: (1) 2 types of antibody (2C3B antibody and 2C5Lantibody) having a recognition sequence within the N-terminal sidefragment polypeptide corresponding to amino acid residue numbers 25 to179 of SEQ ID NO: 1, or forming an immunocomplex with the N-terminalside fragment polypeptide and the FGF-23 full-length protein; (2) 2types of antibody (1D6A antibody and 3C1E antibody) having a recognitionsequence in the C-terminal side fragment polypeptide corresponding toamino acid residue numbers 180 to 251 of SEQ ID NO: 1, and forming animmunocomplex with the C-terminal side fragment polypeptide fragment andthe FGF-23 full-length protein; and (3) an antibody (2A2B antibody) forwhich no immunoprecipitates were detected.

EXAMPLE 11 Detection of FGF-23 Protein by ELISA Using Anti-FGF-23Monoclonal Antibody and Anti-FGF-23 Polyclonal Antibody

2 types of monoclonal antibodies (1D6A antibody and 2C3B antibody) wereseparately immobilized, and then the purified FGF-23 protein wasdetected by a sandwich ELISA system using each of the 5 types ofpolyclonal antibodies (hFGF23-25 antibody, hFGF23-170 antibody,hFGF23-180 antibody, hFGF23-210 antibody, and hFGF23-237 antibody) whoseusefulness as antibody for detection was shown in Example 7.

The above 2 types of monoclonal antibodies purified with a protein Gaffinity column were diluted to 10 μg/ml with a 50 mM sodium hydrogencarbonate solution. 50 μl of the resultant solution was added per wellof a 96-well plate for ELISA (Maxisorp (trademark), Nunc, U.S.A.), andthen incubated at 37° C. for 1 hour for immobilization. Subsequently,the reaction solutions were removed, 50 μl of a blocking solution(SuperBlock (trademark), PIERCE, U.S.A.) was added per well, and theresultant was incubated at room temperature for 30 minutes, therebyperforming blocking. After the solutions were removed, the wells werewashed 3 times with PBS (T-PBS) containing 0.05% Tween 20. 50 μl of eachsolution of the purified FGF-23H protein, purified N-terminal sidefragment polypeptide, or purified C-terminal side fragment polypeptidehaving an His6 tag added thereto, each having a concentration of 0.1μg/ml, was added per well. The solutions were incubated at roomtemperature for 2 hours so as to react with the immobilized antibodies.After antibody reaction, the wells were washed 3 times with T-TBS. 5types of biotin-labeled anti-FGF-23 partial peptide polyclonalantibodies (hFGF23-25 antibody, hFGF23-170 antibody, hFGF23-180antibody, hFGF23-210 antibody, and hFGF23-237 antibody) diluted to 2.5μg/ml with T-TBS containing a 10% blocking solution (Blockace(trademark), DAINIPPON PHARMACEUTICAL CO., LTD. Japan) were incubated atroom temperature for 30 minutes, thereby conducting reaction withsecondary antibodies. After each well was washed 3 times with T-TBS, 50μl of HRP-labeled streptavidin (DAKO, Denmark) diluted 5000-fold withT-TBS containing a 10% blocking solution (Blockace (trademark),DAINIPPON PHARMACEUTICAL CO., LTD. Japan) was added per well. Thesolutions were incubated at room temperature for 30 minutes, so as tobind the streptavidin with biotin-labeled antibodies. Each well waswashed 4 times with T-TBS, 50 μl of tetramethylbenzidine (DAKO,Denmark), which is a peroxidase chromogenic substrate, was added perwell, followed by incubation at room temperature. 7 minutes later, 50 μlof 0.5 M sulfuric acid solution was added per well, so as to stopreaction. Measurement was conducted using a system for measuringabsorbance for a 96-well plate (MTP-300, CORONA ELECTRIC CO., LTD.,Japan), and values were obtained by subtracting absorbance at 570 nmfrom absorbance at 450 nm. The results are shown in FIG. 4. In the caseof the control to which no antibodies had been added, all the valuesobtained by 450 nm-570 nm were 0.015 or less. However, depending on thecombinations of the immobilized antibodies and the antibodies fordetection, reactions specific to the FGF-23 full-length protein (FIG.4A), N-terminal side fragment polypeptide (FIG. 4B), or the C-terminalside fragment polypeptide (FIG. 4C) were observed. Based on differencesin reactivity, the recognition sites of the anti-FGF-23 monoclonalantibodies could be confirmed. Specifically, the full-length FGF-23protein was detected in the case of using a combination of the 2C3Bantibody as an immobilized antibody, and one of 3 types of biotinylatedpolyclonal antibodies (hFGF23-180 antibody, hFGF23-210 antibody, andhFGF23-237 antibody) as antibodies for detection. However, neither theN-terminal fragment polypeptide nor the C-terminal fragment polypeptidewas detected. On the other hand, the full-length FGF-23 protein and theN-terminal side fragment polypeptide were detected strongly by the useof biotin-labeled polyclonal antibodies (hFGF23-25 antibody andhFGF23-170 antibody) recognizing the N-terminal fragment polypeptide,but no C-terminal fragment polypeptide was detected.

Thus, it was confirmed that the 2C3B antibody recognizes the N-terminalfragment polypeptide. When the 1D6A antibody was immobilized, thefull-length protein was detected by the use of polyclonal antibodies asantibodies for detection recognizing the N-terminal fragmentpolypeptides. However, no N-terminal fragments and no C-terminalfragment polypeptides were detected. On the other hand, when thefull-length protein and the C-terminal fragment polypeptides weredetected using polyclonal antibodies recognizing the C-terminal fragmentpolypeptides as antibodies for detection, detection was possible with acombination of the 1D6A antibody with hFGF23-180. These results showedthat the 1D6A antibody competes with the hFGF23-237 polyclonal antibody,so that it was confirmed that the 1D6A antibody recognizes a regionbetween the 237^(th) and the 251^(st) amino acid residues of SEQ ID NO:1.

EXAMPLE 12 Detection of FGF-23 Protein by Sandwich ELISA UsingAnti-FGF-23 Monoclonal Antibodies in Combination

Purified FGF-23 protein was detected by performing sandwich ELISA using4 immobilized types of anti-FGF-23 monoclonal antibodies obtained inExamples 3 and 4, and the 4 types of biotin-labeled anti-FGF-23monoclonal antibodies prepared in Example 6 as antibodies for detection.

4 types of FGF-23 monoclonal antibodies 1D6A antibody, 2A2B antibody,2C3B antibody, and 3C1E antibody) were diluted to 10 μg/ml with a 50 mMsodium hydrogen carbonate solution. 50 μl of the resultant solution wasadded per well of a 96-well plate for ELISA (Maxisorp (trademark), Nunc,U.S.A.), and then incubated at 4° C. for 12 hours for immobilization.Subsequently, the reaction solutions were removed and 50 μl of ablocking solution (SuperBlock (trademark), PIERCE, U.S.A.) was added perwell. The resultant was incubated at room temperature for 20 minutes,thereby performing blocking. After the solutions were removed, the wellswere washed 3 times with TBS containing 0.1% Tween 20(T-TBS). 50 μl ofthe purified FGF-23 protein at a concentration of 0.1 μg/ml was addedper well. The solutions were incubated at room temperature for 1 hour soas to conduct reaction with the immobilized antibodies. After antibodyreaction, the wells were washed 4 times with T-TBS. 3 types ofbiotin-labeled anti-FGF-23 monoclonal antibodies (1D6A antibody, 2C3Bantibody, and 3C1E antibody) diluted at 10 μg/ml with T-TBS containing a10% blocking solution (Blockace (trademark), DAINIPPON PHARMACEUTICALCO., LTD. Japan) were added. The solutions were incubated at roomtemperature for 1 hour, so as to conduct secondary antibody reaction.After each well was washed 4 times with T-TBS, 50 μl of HRP-labeledstreptavidin (DAKO, Denmark) diluted 5000-fold with T-TBS containing a10 % blocking solution (Blockace (trademark), DAINIPPON PHARMACEUTICALCO., LTD. Japan) was added per well. The solutions were incubated atroom temperature for 20 minutes, so as to bind the streptavidin withbiotin-labeled antibodies. Each well was washed 4 times with T-TBS and50 μl of tetramethylbenzidine (DAKO, Denmark), which is a peroxidasechromogenic substrate, was added per well, followed by incubation atroom temperature. 30 minutes later, 50 μl of 0.5 M sulfuric acidsolution was added per well, so as to stop reaction. Measurement wasconducted using a system for measuring absorbance for a 96-well plate(MTP-300, CORONA ELECTRIC CO., LTD., Japan), and values were obtained bysubtracting absorbance at 570 nm from absorbance at 450 nm. The resultsare shown in Table 6. In the case of the control to which immobilizedantibodies or antibodies for detection had not been added, all thevalues obtained by 450 nm-570 nm were 0.033 or less. However, dependingon the combinations of the immobilized antibodies and the antibodies fordetection, increases were observed in absorbance. For example, whensandwich ELISA was performed using as an immobilized antibody the 2C3Bantibody having a recognition site within the range between the 25^(th)and the 179^(th) amino acid residues in the amino acid sequence of theFGF-23 protein represented by SEQ ID NO: 1, and using as antibodies fordetection the biotin-labeled 1D6A and 3C1E antibodies having recognitionsites in the range between the 180^(th) and the 251^(st) amino acidresidues in the amino acid sequence of the FGF-23 protein represented bySEQ ID NO: 1, all the values obtained by 450 nm-570 nm were as high as2.9 or more. Fragmented polypeptides resulting from cleavage between the179^(th) arginine and the 180^(th) serine of the amino acid sequence ofthe FGF-23 protein represented by SEQ ID NO: 1 are excluded from thesubjects to be measured by sandwich ELISA in such a manner. Hence, bythe use of such a combination of antibodies, uncleaved FGF-23 proteincan be detected with high sensitivity without recognizing the N-terminalside polypeptide fragments or the C-terminal side polypeptide fragments.When the full-length FGF-23H protein, the N-terminal side polypeptidefragments, and the C-terminal side polypeptide fragments purified inExample 2 were administered to mice in a manner similar to those ofprevious reports, induction of decreases in serum phosphorus wasobserved only in the case of full-length FGF-23H protein. Thus, it wasrevealed that cleavage between the 179^(th) arginine and the 180^(th)serine of the amino acid sequence of the FGF-23 protein represented bySEQ ID NO: 1 greatly alters the activity of the FGF-23 protein. Themethod shown herein, which involves selectively detecting uncleavedFGF-23 protein while excluding cleaved polypeptides, enables moreprecise detection and measurement of FGF-23 contained within samples andhaving activity. On the other hand, when the 1D6A antibody and the 3C1Eantibody having recognition sites in the region between the 180^(th) andthe 251^(st) amino acid residues in the amino acid sequence of theFGF-23 protein represented by SEQ ID NO: 1 were used as immobilizedantibodies, and the biotin-labeled 1D6A antibody and the 3C1E antibodywere also used as antibodies for detection, it was similarly revealed bycompetition among the antibodies that the group of the 3C1E antibodiesrecognizing the region between the 180^(th) and the 194^(th) amino acidresidues of the amino acid sequence represented by SEQ ID NO: 1 has arecognition site differing from that of the 1D6A antibody recognizingthe region between the 237^(th) and the 251^(st) amino acid residues ofthe amino acid sequence represented by SEQ ID NO: 1. Furthermore, it wasrevealed that the use of these antibodies in combination enablesestablishment of a measurement method with which polypeptide fragmentslocated within the region between the 25^(th) and the 179^(th) aminoacid residues of the amino acid sequence represented by SEQ ID NO: 1 canbe excluded, and C-terminal fragment polypeptides of the 180^(th) to the251^(st) residues can be detected with good sensitivity. TABLE 5Absorbance obtained when FGF-23 protein was detected by sandwich ELISAusing in combination anti-FGF-23 monoclonal antibodies and biotinylatedantibodies thereof as immobilized antibodies and antibodies fordetection, respectively Antibodies for Immobilized antibodies detection1D6A 2C3B 3C1E None 1D6A 0.038 3.469 3.131 0.015 2C3B 1.287 0.466 >3.50.015 3C1E 1.549 >3.5 0.058 0.033 None 0.020 0.024 0.023 0.022

EXAMPLE 13 Quantitative Measurement of FGF-23 Protein by Sandwich ELISAMethod Using Anti-FGF-23 Monoclonal Antibodies

The 2C3B antibodies were diluted to 10 μg/ml with a 50 mM sodiumhydrogen carbonate solution. 50 μl of the resultant solution was addedper well of a 96-well plate for ELISA (Maxisorp (trademark), Nunc,U.S.A.), and then incubated at 4° C. for 12 hours for immobilization.Subsequently, the reaction solutions were removed, 250 μl of a blockingsolution (SuperBlock (trademark), PIERCE, U.S.A.) was added per well andthen the resultant was incubated at room temperature for 30 minutes,thereby performing blocking. After the solutions were removed, the wellswere washed 2 times with TBS (T-PBS) containing 0.1% Tween 20. Asolution was prepared by diluting the FGF-23 protein purified in Example2 at 10, 3, 1, 0.3, 0.1, 0.03, 0.01, or 0.003 ng/ml with T-TBScontaining a 10% blocking solution (Blockace (trademark), DAINIPPONPHARMACEUTICAL CO., LTD. Japan). 50 μl of each solution was added perwell. The solutions were incubated at room temperature for 1 hour, sothat reaction with immobilized antibodies was conducted. After antibodyreaction, the wells were washed 4 times with T-TBS, and then 2 types ofbiotin-labeled anti-FGF-23 monoclonal antibodies (1D6A antibody and 3C1Eantibody) diluted at 10 μg/ml with T-TBS containing a 10% blockingsolution (Blockace (trademark), DAINIPPON PHARMACEUTICAL CO., LTD.Japan) were added. The solutions were incubated at room temperature for30 minutes, thereby performing secondary antibody reaction. After eachwell was washed 4 times with T-TBS, 50 μl of HRP-labeled streptavidin(DAKO, Denmark) diluted 5000-fold with T-TBS containing a 10% blockingsolution (Blockace (trademark), DAINIPPON PHARMACEUTICAL CO., LTD.Japan) was added per well. The solutions were incubated at roomtemperature for 30 minutes, so as to bind the streptavidin withbiotin-labeled antibodies. Each well was washed 4 times with T-TBS, andthen 50 μl of tetramethylbenzidine (DAKO, Denmark), which is aperoxidase chromogenic substrate, was added per well, followed byincubation at room temperature. 25 minutes later, 50 μl of 0.5 Msulfuric acid solution was added per well, so as to stop reaction.Measurement was conducted using a system for measuring absorbance for a96-well plate (MTP-300, CORONA ELECTRIC CO., LTD., Japan), and valueswere obtained by subtracting absorbance at 570 nm from absorbance at 450nm. The results are shown in FIG. 6. Because of the presence of theFGF-23 protein at a concentration of at least 0.03 ng/ml, significantincreases in measured values were observed, and within the concentrationranging from 0.03 to approximately 3 ng/ml, concentration-dependentincreases in values obtained by 450 nm-570 nm were obtained, revealingthat the FGF-23 protein within this concentration range can be detected.

EXAMPLE 14 Preparation of Anti-FGF-23 Monoclonal Antibody-ImmobilizedColumn

A column immobilized anti-FGF-23 antibodies thereto was prepared using acommercial immobilization reagent (SulfoLink (trademark), PIERCE,U.S.A.), which is useful as an antibody column for collecting the FGF-23protein by immunoprecipitation or purifying the FGF-23 protein. 1C3Hantibody, 1D6A antibody, and 2C3B antibody were diluted in a 0.1 Mphosphate buffer (pH 6.0) containing 5 mM EDTA, thereby preparingsolutions at concentrations of 1 mg/ml, 1 mg/ml, and 0.4 mg/ml,respectively. To 1 ml of the antibody solution, 6 mg of2-mercaptoethanol amine was added, the solution was admixed by beingturned upside down at 37° C. for 1 hour, and then disulfide bonds withinthe antibodies were cleaved by reduction reaction. While the buffer wasexchanged with 1.5 ml of a binding buffer (50 mM Tris/HCl, pH 8.5, 5 mMEDTA) solution using an NAP 10 column (Amersham Pharmacia Biotech,U.S.A.), 2-mercaptoethanol amine was removed to stop reduction reaction.These antibody solutions were added to 1 ml of SulfoLink (trademark)coupling gel (PIERCE, U.S.A.) that had been previously washed with abinding buffer. The solutions were admixed by being turned upside downat room temperature for 30 minutes, thereby conducting binding reaction.After centrifugation, resins were washed with a binding buffer. Toperform blocking of unreacted thiol groups, 1 mL of a 0.05 mML-cysteine-HCl solution was added, and then the solutions were admixedby being turned upside down at room temperature for 30 minutes.Subsequently, resins were washed with 1 M NaCl, so as to removeunreacted antibodies and L-cysteine.

EXAMPLE 15 Detection of FGF-23 Protein Existing in the Serum ofCHO-FGF23H Cell-Transplanted Mouse by Immunoprecipitation Method

To examine the state of presence of the FGF-23 protein in blood, cellsexpressing FGF-23H were transplanted into nude mice, and then theFGF-23H protein secreted from these cells into blood were detected by animmunoprecipitation method. 2×10⁷ CHO-FGF-23H cells, or CHO ras clone-1cells as a control were transplanted subcutaneously into each Balb/cnude mouse. On day 32 after transplantation, sera were collected frommice wherein the cells had adhered successfully so as to form tumors.The serum collected from 5 CHO-ras clone-1-transplanted mice, and theserum collected from 6 CHO-FGF23H-transplantated mice were separatelymixed in an equivalent volume. To 150 μl of each mixed serum, 10 μl eachof resins prepared in Example 14 to which the 1D6A antibody had beenimmobilized, resins prepared in Example 14 to which the 2C3B antibodyhad been immobilized, and resins to which no antibodies had beenimmobilized were added. The resultants were admixed by being turnedupside down at 4° C. for 1 hour, thereby performing reaction of theantibodies with FGF-23. The resins were washed 3 times with PBS, so thatunreacted products were removed. 50 μl of a sample buffer (50 mM Tris-ClpH6.8, 1% SDS, 10% glycerol, 0.001% bromophenol blue, 2 mM EDTA, and 20mM DTT) was added to each of the resins. The resultants were heated at95° C. for 5 minutes, and then subjected to centrifugation. The thusobtained supernatants were collected. The supernatants were separated by10%-20% gradient polyacrylamide gel electrophoresis, and then protein ingel was transferred to PVDF membranes (Millipore, U.S.A.) using a SemiDry Blotting System (Owl Separation Systems, U.S.A.). The PVDF membranewas incubated at 4° C. for 12 hours in a solution of biotin-labeled 2A2Bantibodies or 1C3H antibodies that had been diluted at 0.5 μg/ml inBlockace (trademark) (DAINIPPON PHARMACEUTICAL CO., LTD. Japan).Furthermore, HRP-labeled streptavidin (DAKO, Denmark) was allowed toreact with the resultants. The resultants were exposed to film using anECL Plus luminescence system (Amersham Pharmacia Biotech, U.S.A.) for 1hour, and then the film was developed using an automatic processor (FUJIPHOTO FILM CO., LTD., Japan). The results are shown in FIG. 7. For theresins to which no antibodies had been immobilized, no signals wereobserved at all. However, when immunoprecipitation using the 1C3Hantibody and detection using the 2A2B antibody had been conducted, asignal of 22 kDa was detected. When detection had been conducted usingthe 1C3H antibody, signals of 22 kDa and 10 kDa were detected. Inaddition, when immunoprecipitation using the 1D6A antibody and detectionusing the 2A2B antibody had been conducted, a signal of 22 kDa was notdetected, and when detection had been conducted using the 1C3H antibody,a signal of only 10 kDa was detected. Furthermore, whenimmunoprecipitation using the 2C3B antibody and detection using the 2A2Bantibody had been conducted, signals of 22 kDa and 16 kDa were detected,and when detection had been conducted using the 1C3H antibody, a signalof only 22 kDa was detected. It has been shown that the 2A2B antibodyrecognizes the region between the 148^(th) and the 163^(rd) amino acidresidues of SEQ ID NO: 1, and that the 1C3H antibody recognizes theregion between the 180^(th) and the 194^(th) amino acid residues of SEQID NO: 1. Moreover, it has been shown that the 2A2B antibody recognizesthe N-terminal side fragment polypeptide corresponding to the 25^(th)and the 179^(th) amino acid residues of SEQ ID NO: 1 as measured by theWestern blotting method, and that the signal size is 16 kDa as detectedby electrophoresis. It has also been shown that the 1C3H antibodyrecognizes the C-terminal side fragment polypeptide between the 180^(th)and the 251^(st) amino acid residues of SEQ ID NO: 1, and that thesignal size detected is 10 kDa. Thus, it is clear that the signal of 16kDa detected in this experiment indicates the N-terminal side fragmentpolypeptide, and the signal of 10 kDa detected in the same indicates theC-terminal side fragment polypeptide. In addition, the polypeptide of 22kDa collected from the mouse serum can be recognized by the 2C3Bantibody recognizing the N-terminal side region represented by the25^(th) to the 179^(th) amino acid residues, the 2A2B antibodyrecognizing the region between the 148^(th) and the 163^(rd) amino acidresidues, and the antibody produced by 1C3H recognizing the regionbetween the 180^(th) and the 194^(th) amino acid residues, but cannot berecognized by the 1D6A antibody recognizing the region between 237^(th)and the 251^(st) amino acid residues of SEQ ID NO: 1. The signals ofaround 22 kDa observed in this experiment may be generated by thecleavage of the FGF-23 protein, and the cleavage site is present at theC-terminal side from the region that the 1C3H antibody recognizes. Itwas revealed that cleavages and fragments generated therefrom differingfrom cleavage between amino acid numbers 179th arginine and 180th serinerepresented by SEQ ID NO: 1, which we had paid attention to, wereobserved in serum.

EXAMPLE 16 Cleavage of FGF-23 Protein in Serum

(1) Admixture Experiment of Rat Serum and Purified FGF-23 Protein

Cleavage of the FGF-23 protein by serum was examined by admixturingpurified FGF-23 protein with rat serum. Rat serum was added at a finalconcentration of 50% to 20 ng of purified FGF-23 protein. The solutionswere admixed and incubated for 0, 0.5, 1, 2, 4, 8, and 24 hours. Thesolutions were separated by polyacrylamide electrophoresis, and thenprotein in gel was transferred to PVDF membranes (Millipore, U.S.A.)using a Semi Dry Blotting System (Owl Separation Systems, U.S.A.).Western blotting was performed using the 2A2B antibody, so as to detectmetabolites derived from purified FGF-23 protein. The results are shownin FIG. 8. After admixturing with serum, the quantity of the full-lengthFGF-23 protein existing in the solution decreased as the incubation timeincreased, and the appearance and increase of new fragments of 22kDawere observed.

(2) Admixture Experiment of Human Serum or Plasma with Purified FGF-23Protein

Human serum or human plasma was added at a final concentration of 50 %to 20 ng of purified FGF-23 protein, followed by incubation at 37° C.for 3 hours. The solutions were separated by polyacrylamideelectrophoresis, and then protein in gel was transferred to PVDFmembranes (Millipore, U.S.A.) using a Semi Dry Blotting System (OwlSeparation Systems, U.S.A.). Western blotting was performed using the2A2B antibody, so as to detect metabolites derived from purified FGF-23protein. The results are shown in FIG. 9. When human serum and theFGF-23 protein were mixed, a band of 22 kDa appeared. However, whenhuman plasma and the FGF-23 protein were mixed, a band of 22 kDa was notobserved. Therefore, the band of 22 kDa was assumed to result fromprotein cleavage enzymes existing in the serum.

EXAMPLE 17 Identification of Enzyme Cleaving FGF-23 Protein

(1) Cleavage of FGF-23 by Thrombin

Thrombin (Sigma, U.S.A.) was added at a final concentration of 1 unit/mlto 20 ng of purified FGF-23 protein, followed by 3 hours of incubation.The solution was separated by polyacrylamide electrophoresis, and thenprotein in gel was transferred to a PVDF membrane (Millipore, U.S.A.)using a Semi Dry Blotting System (Owl Separation Systems, U.S.A.).Western blotting was performed using the 2A2B antibody, so as to detectmetabolites derived from purified FGF-23 protein. The results are shownin FIG. 10. The full-length FGF-23 protein disappeared, and the bandthereof was converted to a band of 22 kDa.

(2) Action of Hirudine to Inhibit Cleavage of FGF-23 Protein by Serum

For the purpose of examining the involvement of thrombin in serum incleavage of FGF-23 by serum, the effect of hirudine, which is known as athrombin selective inhibitor, was examined. Rat serum was added at afinal concentration of 50% to 20 ng of purified FGF-23 protein, followedby 4 hours of incubation. Furthermore, at the time of addition of ratserum, hirudine was added at 1.0 unit/ml, followed by incubationsimilarly. The solution was separated by polyacrylamide electrophoresis,and then protein in gel was transferred to a PVDF membrane (Millipore,U.S.A.) using a Semi Dry Blotting System (Owl Separation Systems,U.S.A.). Western blotting was performed using the 2A2B antibody, so asto detect metabolites derived from purified FGF-23 protein. The resultsare shown in FIG. 11. By the addition of rat serum, some of the FGF-23proteins were converted into polypeptides of 22 kDa, and co-existencewith hirudine suppressed the appearance of a band of 22 kDa. Thus, itwas shown that the cleavage of the FGF-23 protein generated by serum isdue to thrombin or enzymes analogous thereto.

EXAMPLE 18 Identification of Cleavage Site of FGF-23 Protein by Serum

To identify the cleavage site of FGF-23 generated by serum, 10 μg ofpurified FGF-23 protein and rat serum were admixed for 24 hours, therebygenerating polypeptides of 22 kDa. The polypeptides of 22 kDa in thissolution were adsorbed to the anti-1C3H antibody column prepared inExample 14, eluted, and purified. The antibody column used herein hadbeen prepared by adsorbing 200 μg of biotinylated 1C3H antibodies on 500μl of streptavidin-immobilized resins (Amersham Pharmacia Biotech,U.S.A.). The FGF-23-derived protein was eluted from the antibody columnusing a 0.1 M glycine solution (pH 2.7). The thus purified polypeptideswere separated by SDS-polyacrylamide gel electrophoresis and stained byCBB staining, so that the purified polypeptide of 22 kDa was identifiedas shown in FIG. 12. This band was excised and subjected to MALDI-TOF MSanalysis. Based on the molecular weight obtained from this analysis, thepolypeptide of 22 kDa was shown to have a sequence ranging from the25^(th) tyrosine to the 196^(th) arginine of the amino acid sequencerepresented by SEQ ID NO: 1.

These results revealed that in serum, the FGF-23 protein is cleaved at aposition following the 196^(th) arginine of the amino acid sequencerepresented by SEQ ID NO: 1 by thrombin contained in serum, and thenconverted to a polypeptide represented by a sequence between the 25^(th)and the 196^(th) amino acid residues of SEQ ID NO: 1. As describedabove, measurement of FGF-23 protein having activity in blood requiresdetection of polypeptides uncleaved between the 179^(th) and the180^(th) amino acid residues of the amino acid sequence of SEQ ID NO: 1.It was shown that the full-length FGF-23 protein having activity ispartially cleaved between the 196 ^(th) and the 197 ^(th) amino acidresidues during the process of the preparation of a serum sample, andthe anti-FGF-23 monoclonal antibody that recognizes the C-terminuscannot recognize the metabolite thereof. Specifically, when sandwichELISA was conducted using a combination of an antibody recognizing theregion between the 25^(th) and the 179^(th) amino acid residues of theamino acid sequence of SEQ ID NO: 1, and an antibody recognizing theregion between the 180^(th) and the 196^(th) amino acid residues of theamino acid sequence of SEQ ID NO: 1, the effect of cleavage resultingfrom serum preparation can be ignored. However, when an antibodyrecognizing the region between the 25^(th) and the 179^(th) amino acidresidues of the amino acid sequence of SEQ ID NO: 1, and an antibodyrecognizing the region between the 197^(th) and the 251^(st) amino acidresidues of the amino acid sequence of SEQ ID NO: 1 were used, measuredvalues in serum may decrease compared with those in plasma. Among theanti-FGF-23 monoclonal antibodies obtained herein, the 1C3H antibody andthe 3C1E antibody recognize the region between the 180^(th) and the194^(th) amino acid residues of the amino acid sequence represented bySEQ ID NO: 1. Thus, a 22 kDa protein (corresponding to the 25^(th) andthe 196^(th) amino acid residues of the amino acid sequence of SEQ IDNO: 1) produced by the cleavage of FGF-23 in serum can be recognized, asdistinct from polypeptide fragments generated by the cleavage betweenthe 179^(th) and the 180^(th) amino acid residues of the amino acidsequence of SEQ ID NO: 1. Thus, it was revealed that these antibodiesare very useful in producing a method for measuring serum samples.

EXAMPLE 19 Quantitative Analysis of FGF-23 Protein Concentrations inSerum and Plasma of Patients with Tumor-Induced Osteomalacia

It has been reported that in tumor-induced osteomalacia, FGF-23 isexcessively produced in tumors, and it is known that the disease iscured by removing the causative tumors. However, the causative tumor oftumor-induced osteomalacia is generally small because of its low growthability and is often difficult to discern. Thus, diagnosisdifferentiating between tumor-induced osteomalacia and otherhypophosphatemic diseases is difficult. Thus, if it can be demonstratedthat blood FGF-23 concentrations can be an indicator for diagnosingtumor-induced osteomalacia, and a method by which the blood FGF-23concentrations can be quantitatively measured is developed, such amethod is clinically useful.

Serum and plasma samples were obtained from tumor-induced osteomalaciapatients, and quantitative analysis of FGF-23 in the analytes wasattempted using a sandwich ELISA system using anti-FGF-23 monoclonalantibodies. The ELISA system using anti-FGF-23 monoclonal antibodies wasperformed using the above method. The 2C3B antibody was used as animmobilized antibody, and the biotin-labeled 3C1E or 1D6A antibody wasused as an antibody for detection. The purified 2C3B antibodies werediluted to 10 μg/ml with a 50 mM sodium hydrogen carbonate solution. 50μl of the resultant solution was added per well of a 96-well plate forELISA (Maxisorp (trademark), Nunc, U.S.A.), and then incubated at 4° C.for 12 hours for immobilization. Subsequently, the reaction solutionswere removed, 250 μl of a blocking solution (SuperBlock (trademark),PIERCE, U.S.A.) was added per well, and the resultant was incubated atroom temperature for 30 minutes, thereby performing blocking. After thesolutions were removed, the wells were washed 2 times with TBS (T-TBS)containing 0.1% Tween 20. To avoid non-specific reaction to mouseantibodies, the serum and plasma samples of the tumor-inducedosteomalacia patients were diluted 2-fold with T-TBS solutionscontaining mouse IgG1 at a concentration of 80 μg/ml as isotypecontrols. Furthermore, to confirm specific reaction to the FGF-23protein, the samples were diluted 2-fold with T-TBS containing anexcessive volume (80 μg/ml) of the 2C3B antibody that had been used forimmobilization. Standard solutions to produce a calibration curve wereprepared by adding purified FGF-23 protein to serum or plasma of normalsubjects diluted with a solution similarly containing the isotypecontrol antibodies to result in concentrations of 0.5, 0.25, 0.125,0.061, 0.031, and 0.015 ng/ml, respectively. 50 μl of each sample wasadded per well of a microtiter plate, and then the solutions wereincubated at room temperature for 1 hour, thereby performing reaction ofthe FGF-23 protein in the analytes with immobilized antibodies. Afterantibody reaction, the wells were washed 4 times with T-TBS, and then 2types of biotin-labeled anti-FGF-23 monoclonal antibodies (1D6A antibodyand 3C1E antibody) diluted at 10 μg/ml with T-TBS containing a 10%blocking solution (Blockace (trademark), DAINIPPON PHARMACEUTICAL CO.,LTD. Japan) were added. The solutions were incubated at room temperaturefor 30 minutes, thereby performing secondary antibody reaction. Aftereach well was washed 4 times with T-TBS, 50 μl of HRP-labeledstreptavidin (DAKO, Denmark) diluted 5000-fold with T-TBS containing a10% blocking solution (Blockace (trademark), DAINIPPON PHARMACEUTICALCO., LTD. Japan) was added per well. The solutions were incubated atroom temperature for 30 minutes, so as to bind the streptavidin withbiotin-labeled antibodies. Each well was washed 4 times with T-TBS, andthen 50 μl of tetramethylbenzidine (DAKO, Denmark), which is aperoxidase chromogenic substrate, was added per well, followed byincubation at room temperature. 30 minutes later, 50 μl of 0.5 Msulfuric acid solution was added per well, so as to stop reaction.Measurement was conducted using a system for measuring absorbance for a96-well plate (MTP-300, CORONA ELECTRIC CO., LTD., Japan), and valueswere obtained by subtracting absorbance at 570 nm from absorbance at 450nm. A value representing an FGF-23-specific reaction was obtained bysubtracting an A450-A570 value measured in the presence of an excessivequantity of the 2C3B antibody from an A450-A570 value measured in thepresence of an excessive quantity of the isotype control antibody. Theresults are shown in FIG. 13. When 1D6A was used as an antibody fordetection, measured values of the plasma sample and the serum samplewere 0.033 and 0.008, respectively. The measured value was clearly lowerfor the serum sample than for the plasma sample. On the other hand, when3C1E was used as an antibody for detection, the measured value of theserum sample and that of the plasma sample were almost the same. Theseresults suggested that the FGF-23 protein might be partially cleaved inthis sample, as cleaved in serum described in Example 18. Thus, it wasrevealed that measured values of clinical samples differ depending ondifferences in antibodies for detection as described above. Hence,sandwich ELISA was conducted using as an antibody for detection the 3C1Eantibody with which cleaved products generated upon serum preparationcan also be measured, and then FGF-23 concentrations in plasma samplescollected before and after tumorectomy of the patients were measured.Using standard solutions prepared by adding purified FGF-23 protein atvarious concentrations to plasma samples of normal subjects, acalibration curve was produced based on increases in absorbancecorresponding to added quantities of purified protein. The thus producedcalibration curve is shown in FIG. 14A. The thus obtained calibrationcurve can also be used to quantify FGF-23 protein in a human plasma orserum sample diluted 2-fold by detecting the concentration thereofcorresponding to that within a range between 30 pg/ml and at leastapproximately 500 pg/ml of the FGF-23 protein in the standard solutionsas a significantly increased concentration. Under such conditions, theFGF-23 concentrations in plasma samples collected the day beforetumorectomy and collected 1 month or more after tumorectomy fromtumor-induced osteomalacia patients were quantitatively measured. Theresults are shown in FIG. 14B. FGF-23 protein was present at aconcentration of approximately 270 pg/ml in the plasma samples collectedbefore tumorectomy. However, in the samples collected after tumorectomy,the FGF-23 protein concentrations decreased to detection sensitivity (30pg/ml) or less. Thus, it was clearly shown that in tumor-inducedosteomalacia, FGF-23 is present at a measurable concentration in bloodbecause of the presence of causative tumors, and the blood FGF-23concentrations significantly decrease due to the removal of the tumors.Accordingly, it was shown that ELISA using anti-FGF-23 monoclonalantibodies is useful in diagnosis of tumor-induced osteomalacia, and bythe use of antibodies whose recognitions sites are specified,quantitative measurement is possible while taking into consideration theeffect of cleavage of FGF-23 by serum.

EXAMPLE 20 Immunohistological Staining Using Anti-FGF-23 MonoclonalAntibody

The causative tumors of tumor-induced osteomalacia wereimmunohistologically stained using an anti-FGF-23 monoclonal antibody.An excised tumor was immersed in an embedding solution for thepreparation of frozen sections, and then frozen using acetone cooledwith liquid nitrogen, thereby preparing blocks of the frozen tumor. Inaddition, as a control, a frozen block was also similarly prepared fromtissue thought to be normal that had been excised upon tumorectomy fromthe periphery of the tumor tissue. The frozen blocks were thinly slicedat a thickness of 4 μm using a cryostat (CM1900, LEICA, Germany). Thesection was made to adhere to a MAS coat slide glass (MATSUNAMI GLASSIND., LTD., Japan) while being cooled and dried. The thus preparedfrozen sections were stored at −20° C. The slide glasses to which thefrozen sections had adhered were incubated in acetone at roomtemperature for 5 minutes, so that the tissues were immobilized on theslide glasses. After washing with PBS, incubation was performed in PBScontaining 1% hydrogen peroxide and 0.1% sodium azide at roomtemperature for 30 minutes, thereby inactivating endogenous peroxidase.Subsequently, the resultants were washed with PBS containing 0.1%Tween20, and then incubation was performed in PBS containing 4% skimmilk at room temperature for 30 minutes, thereby performing blocking.Next, incubation was performed with PBS containing 10 μg/mlbiotin-labeled 1C3H and 2C3B antibodies at room temperature for 1 hour,thereby causing FGF-23 in the tissue to react with added monoclonalantibodies. After washing with PBS containing 0.1% Tween20, horseradishperoxidase was bound to the biotin-labeled monoclonal antibodiesspecifically bound to the tissue sections using a peroxidase kit(Vectastain Elite ABC (trademark), VECTOR Laboratories, U.S.A.). Afterwashing with PBS containing 0.1% Tween20, reaction of peroxidase withsubstrates (DAKO liquid DAB substrate-chromogen system (trademark),DAKO, Denmark) was conducted, and then reaction was stopped using PBS.After washing with ion exchanged water, incubation was performed in asolution prepared by diluting 5-fold Hematoxylin Mayer (Merck & Co.,U.S.A.) with ion exchanged water at room temperature for 30 seconds,followed by washing in running water. Subsequently, dehydration wasperformed using ethanol, penetration was performed using xylene, andthen the resultants were sealed using an encapsulation solution. Theresults are shown in FIG. 15. The sites thought to have reacted withFGF-23 in tumor tissues were stained brown. However, no such stainingimages were observed in tissues at the periphery of the tumor.Accordingly, the presence of FGF-23 at high concentrations was confirmedin the causative tumors of tumor-induced osteomalacia.

EXAMPLE 21 Expression and Purification of Mouse FGF-23 Protein

A mouse FGF-23 sequence has already been reported (Yamashita, T., et al.Biochem. Biophys. Res. Commun. 277: 494-498, 2000). The sequenceinformation of the mouse FGF-23 can be obtained by searching the NCBIgene sequence database based on this sequence. Based on the thusobtained sequence information, nested PCR was performed to obtain cDNAencoding the mouse FGF-23 protein. As primers to be used for the firststage of amplification reaction, an mF5 primer (SEQ ID NO: 25) and anmF3 primer (SEQ ID NO: 26) having sequences complementary to the 5′ andthe 3′ untranslated regions, respectively, of the cDNA sequence of mouseFGF-23 were synthesized. After keeping the temperature at 94° C. for 1minute, 30 cycles of PCR was performed using cDNA (Clontech, U.S.A.)derived from mouse lungs as a template and LA-Taq DNA polymerase (TAKARASHUZO, Japan), each cycle consisting of 94° C. for 30 seconds, 55° C.for 30 seconds, and 72° C. for 45 seconds. Next, the second stage ofreaction was performed using an mF23F primer represented by SEQ ID NO:27 containing a region from the initiation codon to the translatedregion, and an mF23R primer represented by SEQ ID NO: 28 containing aregion from the translated region to the termination codon. In the mF23Fprimer, an EcoR I restriction enzyme recognition sequence for cloningand a Kozak sequence had been introduced. In the meantime, in the mF23Rprimer, a Not I restriction enzyme recognition sequence had beenintroduced following the termination codon. After the temperature waskept at 98° C. for 1 minute, 35 cycles of PCR were performed using thereaction solution in the 1^(st) stage as a template and PyroBest DNApolymerase (TAKARA SHUZO, Japan), each cycle consisting of 98° C. for 10seconds, 58° C. for 10 seconds, and 72° C. for 60 seconds. Thus, thecDNA sequence encoding the mouse FGF-23 protein was amplified. The cDNAwas cloned into the EcoR I site and the Not I site of a pEAK8 vector(Edge Biosystem, U.S.A.). This vector is referred to as pEAK8mFGF-23.Furthermore, according to the method for preparing FGF-23RQH shown inExample 1, mouse FGF-23RQ was constructed wherein a mutation had beenintroduced so as to substitute an arginine residue at a cleavage enzymerecognition site of mouse FGF-23 with a glutamine residue. The mutationwas introduced according to the method described in Example 1(3). Forthe introduction of the mutation, a forward mRQF primer (SEQ ID NO: 29)and a reverse mRQR primer (SEQ ID NO: 30) were synthesized. First, PCRwas performed using the pEAK8mFGF-23 as a template, a combination of themF23F primer and the mRQR primer, and a combination of the mF23R primerand the mRQF primer, thereby obtaining PCR fragments from each case.Next, PCR was performed using a mixture of both products as a template,and the mF23F primer and the mF23R primer which were located on bothends of mouse FGF-23 cDNA, thereby amplifying the mouse FGF-23 cDNAwherein the mutation had been introduced. The product was cloned intothe EcoR I restriction enzyme site and the Not I restriction enzyme siteof a pEAK8/IRES/EGFP plasmid. The resultant was purified and then thenucleotide sequence was determined, confirming that the mouse FGF-23cDNA having the target mutation introduced therein had been successfullycloned. This is referred to as pEAK8/IRES/EGFP/mFGF-23RQ. Furthermore,this plasmid was introduced into CHO ras clone-1, 5 μg/ml puromycin(Sigma, U.S.A.) was added, and then drug-resistant cells were selected.After cloning, mouse FGF-23-expressing cells were obtained. Such cellsare referred to as CHO-mFGF23RQ. The CHO-mFGF23RQ cells were cultured ina roller bottle, thereby obtaining approximately 12 liters of culturesupernatant. The resultant was subjected to purification according tothe method for purifying the FGF-23 protein described in Example 2(3),thereby obtaining purified mouse FGF-23RQ. mF5:ATTAGCCACTCAGTGCTGTGCAATG (SEQ ID NO: 25) mF3: GCAGCCTGGCCTGGGGACCTA(SEQ ID NO: 26) mF23F: GGAATTCCACCATGCTAGGGACCTGCCTTAGA (SEQ ID NO: 27)CTC mF23R: ATAGTTTAGCGGCCGCCTAGACGAACCTGGGAAA (SEQ ID NO: 28) GGGGCGACAmRQF: TTCGCCCACGGCAACACACGCAAAGCGCCGAG (SEQ ID NO: 29) GAC mRQR:GTCCTCGGCGCTTTGCGTGTGTTGCCGTGGGC (SEQ ID NO: 30) GAA

EXAMPLE 22 Cross Reactivity of Anti-Human FGF-23 Monoclonal Antibodiesand FGF-23

The purified mouse FGF-23RQ protein was separated by SDS-polyacrylamidegel electrophoresis, transferred to PVDF membranes (Millipore, U.S.A.),and then subjected to Western blotting using the 3C1E antibody. Thus, aband was detected at approximately 32.5 kDa as shown in FIG. 16A. Thepurified mouse FGF-23RQ protein solution was serially diluted, and thendetection was attempted by sandwich ELISA using anti-human FGF-23monoclonal antibodies. The concentrations of the mouse FGF-23RQ proteinsolution used herein were not precisely obtained. As determined from thedepth of bands detected when the solutions were subjected to separationby SDS-polyacrylamide gel electrophoresis while being aligned with thepurified human FGF-23 protein having a known concentration, and CBBstaining, it was considered that the concentrations of the mouseFGF-23RQ protein solutions were within the range between 1 and 5 μg/ml,when expressed using a relative value of 10 as in FIG. 16B. Using theimmobilized 2C3B antibody and the 3C1E antibody as an antibody fordetection, the diluted mouse FGF-23RQ protein solutions as testsubstances were measured. The results are shown in FIG. 16B. Since 450nm-570 nm values were observed to increase in a manner depending on theconcentrations of the purified mouse FGF-23RQ protein, it became clearthat the mouse FGF-23 protein can be detected in aconcentration-dependent manner by ELISA using the anti-human FGF-23monoclonal antibodies used herein. Furthermore, it could be confirmedthat anti-human FGF-23 monoclonal antibodies can recognize the mouseFGF-23 protein, suggesting that the anti-human FGF-23 monoclonalantibodies observed in Examples 27 and 28 act to neutralize or modifythe activity by binding with mouse endogenous FGF-23.

EXAMPLE 23 Detection of Blood FGF-23 Protein of HereditaryHypophosphatemia Model Mouse

FGF-23 is known to be a factor inducing tumor-induced osteomalacia, andis also known to be involved in inducing ADHR because of missensemutations found in the FGF-23 gene in ADHR. However, in another form ofhereditary hypophosphatemia, XLH, although the responsible gene has beenelucidated, the mechanism inducing the disease has not yet beenunderstood well. In addition, the relationship with FGF-23 is not known.For the purpose of examining the involvement of FGF-23 in this disease,measurement of blood FGF-23 concentrations in Hyp mice (the XLH modelmice) was attempted.

ELISA was performed using the 2C3B antibody as an immobilized antibodyand the biotin-labeled 3C1E antibody as an antibody for detection. Thepurified 2C3B antibody was diluted to 10 μg/ml with a 50 mM sodiumhydrogen carbonate solution. 50 μl of the resultant solution was addedper well of a 96-well plate for ELISA (Maxisorp (trademark), Nunc,U.S.A.), and then incubated at 4° C. for 12 hours for immobilization.Subsequently, the reaction solutions were removed, 250 μl of a blockingsolution (SuperBlock (trademark), PIERCE, U.S.A.) was added per well,and then the resultant was incubated at room temperature for 30 minutes,thereby performing blocking. After the solutions were removed, the wellswere washed 2 times with TBS (T-PBS) containing 0.1% Tween 20. Blood wascollected from the eye sockets of 27- to 30-week-old Hyp mice and normalcontrol mice obtained from the same brood, and then serum samples wereprepared therefrom. The serum samples were diluted 2-fold with T-TBScontaining the 2C3B antibody or a mouse IgG1 antibody as an isotypecontrol, at a concentration of 40 μg/ml. 50 μl of each sample was addedper well of a microtiter plate, and then the samples were incubated atroom temperature for 1 hour, thereby causing the FGF-23 protein in theanalytes to react with the immobilized antibodies. After antibodyreaction, the wells were washed 4 times with T-TBS, and then the 3C1Eantibody diluted to 2 μg/ml with T-TBS containing a 10% blockingsolution (Blockace (trademark), DAINIPPON PHARMACEUTICAL CO., LTD.Japan) was added. The solutions were incubated at room temperature for30 minutes, thereby performing secondary antibody reaction. After eachwell was washed 4 times with T-TBS, 50 μl of HRP-labeled streptavidin(DAKO, Denmark) diluted 5000-fold with T-TBS containing a 10% blockingsolution (Blockace (trademark), DAINIPPON PHARMACEUTICAL CO., LTD.Japan) was added per well. The solutions were incubated at roomtemperature for 30 minutes, so as to bind the streptavidin withbiotin-labeled antibodies. Each well was washed 4 times with T-TBS, andthen 50 μl of tetramethylbenzidine (DAKO, Denmark), which is aperoxidase chromogenic substrate, was added per well, followed byincubation at room temperature. 30 minutes later, 50 μl of 0.5 Msulfuric acid solution was added per well, so as to stop reaction.Measurement was conducted using a system for measuring absorbance for a96-well plate (MTP-300, CORONA ELECTRIC CO., LTD., Japan), and values(A450-A570) were obtained by subtracting absorbance at 570 nm fromabsorbance at 450 nm. A value showing a FGF-23-specific reaction wasobtained by subtracting an A450-A570 value obtained by the addition ofthe 2C3B antibody used as an absorption antibody from an A450-A570 valueobtained by the addition of the isotype control antibody upon reaction.The results are shown in FIG. 17. The results shown in FIG. 17 clearlyshow significantly elevated blood FGF-23 concentrations of Hyp mice.Since Hyp mice develop hypophosphatemia due to a PHEX gene deficiency,and XLH, the human hypophosphatemia, is caused by mutation or deficiencyin the PHEX gene, Hyp mice are thought to be XLH model mice. Thisresults from the strong suggestion that an elevated blood FGF-23concentration is present as a factor inducing hyphosphatemia in XLH.

EXAMPLE 24 Mutual Control Action of FGF-23 and 1,25D

(1) Decrease in 1,25D by Administration of FGF-23H

5 μg per mouse of the purified FGF-23H protein was administered via thecaudal vein to six 6-week-old BALB/c male mice. At 1, 4, and 9 hoursafter administration, blood was collected, and then blood 1,25Dconcentrations were measured. The results are shown in FIG. 18A. At 3hours after administration of FGF-23H, a significant decrease wasobserved in 1,25D, and the concentration was observed to furtherdecrease at 9 hours after the administration.

(2) Induction of FGF-23 by Administration of 1,25D

1,25D was administered to mice, and changes in blood FGF-23 wereexamined. Six 7-week-old BALB/c male mice composed each group. A groupto which 0.025 μg of 1,25D dissolved in PBS containing 0.05% tween wasadministered, and a control group to which PBS containing 0.05% tween,the vehicle, was administered were established. Administration wasperformed intraperitoneally. 8 hours after the administration, blood wascollected from the heart under anesthesia, and then serum samples wereprepared. These samples were subjected to ELISA by the same method asthat of Example 23, and then the quantities of FGF-23 in blood weremeasured using the calibration curve prepared using the standardsolutions of the human FGF-23 protein. The results are shown in FIG.18B. At 8 hours after the administration of 1,25D, a significantincrease was observed in blood FGF-23.

Accordingly, it was shown that FGF-23 has a close mutual controlrelationship with 1,25D.

EXAMPLE 25 Blood FGF-23 Concentration in Renal Failure HyperphosphatemiaModel

CE-2 (CLEA Japan, Inc., Japan) mixed with adenine at the rate of 0.75%was fed to 7-week-old Wistar rats, preparing a renal failurehyperphosphatemia model. To a control group, CE-2 was fed. 3 weeks afterthe start of feeding the mixed feed, blood was collected via the caudalartery, so as to collect serum. After blood collection, rats were housedin metabolic cages and urine was collected for 24 hours. Serum phosphateconcentrations were measured. The results are shown in FIG. 19A.Creatinine in serum and that in urine were measured by an enzyme methodusing a commercial kit (CRE-EN Kainos (trademark), KAINOS LABORATORIES,INC., Japan). The results are shown in FIG. 19B. Moreover, serum FGF-23concentrations were measured using an ELISA system using the 2C3Bantibody as an immobilized antibody and the 3C1E antibody as an antibodyfor detection. The results are shown in FIG. 19C.

That a disorder of renal functions had progressed in this model wasclearly shown by decreased urine creatinine concentrations and increasedblood creatinine concentrations. With this progress, the model developedhyperphosphatemia. In this model, blood FGF-23 protein was at asignificantly high level. This model was thought to reflect a mode ofrenal failure, suggesting the possibility that FGF-23 acts as a factorinducing some complications in decreased renal functions and inhemodialysis patients.

EXAMPLE 26 Detection by Immunoprecipitation Method of FGF-23 ProteinExisting in the Plasma of Tumor-Induced Osteomalacia Patients

To examine a state of the presence of FGF-23 protein in blood, detectionwas performed by the immunoprecipitation method using plasma of normalsubjects or plasma of tumor-induced osteomalacia patients. To 400 μl ofa mixed plasma sample, 20 μl of resins (prepared in Example 14) to whichthe 2C3B antibody had been immobilized, or resins to which no antibodieshad been immobilized, was added. The resultants were admixed by beingturned upside down at 4° C. for 1 hour, so as to cause the antibodies toreact with FGF-23. Subsequently, the resins were washed 4 times withPBS, thereby removing unreacted products. 50 μl of a sample buffer (50mM Tris-Cl pH6.8, 1% SDS, 10% glycerol, 0.001% bromophenol blue, 2 mMEDTA, and 20 mM DTT) was added to each type of resin. After heating at95° C. for 5 minutes, centrifugation was performed, and then theobtained supernatants were collected. The supernatants were separated by10-20% gradient polyacrylamide gel electrophoresis, and then protein ingel was transferred to PVDF membranes (Millipore, U.S.A.) using a SemiDry Blotting System (Owl Separation Systems, U.S.A.). Then, the PVDFmembrane was incubated at room temperature for 2 hours in a solution ofbiotin-labeled 3C1E antibody that had been diluted at 0.5 μg/ml in T-TBS(Sigma, U.S.A.). Furthermore, HRP-labeled streptavidin (DAKO, Denmark)was allowed to react with the solutions. The resultants were exposed tofilm using an ECL Plus luminescence system (Amersham Pharmacia Biotech,U.S.A.) for 1 hour, and then the film was developed using an automaticprocessor (FUJI PHOTO FILM CO., LTD., Japan). The results are shown inFIG. 20. For the resins to which no antibodies had been immobilized,only plasma-derived non-specific signals were observed. However,regarding the products immunoprecipitated using the 2C3B antibody, aband was detected at 32 kDa, and was suspected to represent thefull-length FGF-23 in the plasma of tumor-induced osteomalacia patients.Furthermore, no bands of 22 kDa corresponding to products cleaved bythrombin were detected at all. In the meantime, in normal subjects,neither 22 kD band nor 32 kD band was observed. From these results, itwas revealed that FGF-23 expressing at high levels in tumor-inducedosteomalacia exists in its full-length form without being cleaved inblood.

EXAMPLE 27 Experiment of Administration of Anti-Human FGF-23 MonoclonalAntibodies to Normal Mice

To examine the effect of the anti-human FGF-23 monoclonal antibodies onnormal mice, the following experiment was conducted. Normal mice(BALB/c, male, 12-week-old) were randomly divided into 5 groups, each ofwhich consisted of 4 mice. As shown in FIG. 21A, single administrationof 0.15 ml of PBS as a vehicle was performed via the caudal vein permouse of group 1, that of 0.67 mg/ml anti-human FGF-23 monoclonalantibodies (1D6A antibody, 2C3B antibody, and 3C1E antibody) wasperformed via the caudal vein per mouse of groups 2, 3, and 4,respectively, and that of 0.67 mg/ml anti-TPO monoclonal antibodies as acontrol was performed via the caudal vein per mouse of group 5. 24 hoursafter the administration, blood was collected from the heart under etheranesthesia, and then serum was separated using a microtainer (BectonDickinson, U.S.A.). Phosphate concentrations in the thus obtained serumwere measured using P (phosphorus)-Test Wako (Wako Pure ChemicalIndustries, Ltd., Japan), the serum calcium concentrations were measuredusing a Calcium-Test Wako (Wako Pure Chemical Industries, Ltd., Japan),and serum 1,25D concentrations were measured using a 1,25(OH)2D RIA KitTFB (TFB, U.S.A.) according to the attached documents. During the periodfrom administration to blood collection, each group of mice was kept ina plastic cage, and fed ad libitum with CE-2 solid feed (CLEA Japan,Inc., Japan) containing 1% phosphorus and 1% calcium, along with tapwater.

The results are shown in FIG. 21A. Measured values are expressed withaverage value±standard deviation of each group. Groups marked with a “*”symbol showed p<0.01 for both the group to which the vehicle (PBS) hadbeen administered and the group to which anti-TPO antibody had beenadministered when tests of significance were conducted by Student-t.

EXAMPLE 28

In Example 27, although increases were observed in serum phosphorusconcentrations in the case of the 3C1E antibody, decreases were observedin serum 1,25D concentrations. However, it is known that blood 1,25Dconcentrations change rapidly, such that when a 1,25D concentration istemporarily increased, it can exhibit a lower concentration thereafter.Hence, an experiment of administration of the 3C1E antibody wasconducted again. The 3C1E antibody was administered intravenously at 400μg/head to 6 normal mice. 8 hours after the administration, serum 1,25Dconcentrations were measured. As a control, serum 1,25D concentrationsof mice similarly treated by administration of PBS were measured. Theresults are shown in FIG. 21B. In this experiment, significant increasesresulting from the administration of the 3C1E antibody were observed inserum 1,25D concentrations.

EXAMPLE 29 Effect of Anti-FGF-23 Antibody (3C1E Antibody) to Recover1,25(OH)₂D in Rats Having Renal Failure Induced by Adenine

As described in Example 25, in rats having renal failure induced byadenine, on week 3 after the production of the model, significantincreases were observed in serum phosphorus concentrations and bloodFGF-23 concentrations. Furthermore, in this model, already on day 7after administration of adenine, decreases were also observed in blood1,25D concentrations as renal functions decreased (FIG. 22A). Hence, tocompare and examine blood FGF-23 concentrations in the early stage of adisorder of renal functions, quantification was conducted in the mannerdescribed below.

In ELISA assay, the 2C3B antibody was used as an immobilized antibody,and the biotin-labeled 3C1E antibody was used as an antibody fordetection. First, the purified 2C3B antibody was diluted to 5 μg/ml witha 50 mM sodium hydrogen carbonate solution. 50 μl of the resultantsolution was added per well of a 96-well plate for ELISA (Maxisorp(trademark), Nunc, U.S.A.), and then incubated at 4° C. for 12 hours forimmobilization. Subsequently, the reaction solutions were removed, 250μl of a blocking solution (SuperBlock (trademark), PIERCE, U.S.A.) wasadded per well, and then the resultant was incubated at room temperaturefor 30 minutes, thereby performing blocking. After the solutions wereremoved, the wells were washed 2 times with TBS (T-TBS) containing 0.1%Tween 20. The serum was prepared and diluted 2-fold with T-TBS. Inaddition, standard products were diluted using rat serum that had beensubjected to the anti-2C3B-antibody-immobilized resins prepared inExample 14 so as to remove endogenous FGF-23, and then diluted 2-foldwith T-TBS. 50 μl of each sample was added per well of a microtiterplate, and then the samples were incubated at room temperature for 1hour, thereby causing the FGF-23 protein in the analytes to react withthe immobilized antibodies. After antibody reaction, the wells werewashed 4 times with T-TBS, and then the 3C1E antibody diluted to 1.5μg/ml with T-TBS containing a 10% blocking solution (Blockace(trademark), DAINIPPON PHARMACEUTICAL CO., LTD. Japan) were added. Thesolutions were incubated at room temperature for 30 minutes, therebyperforming secondary antibody reaction. After each well was washed 4times with T-TBS, 50 μl of HRP-labeled streptavidin (DAKO, Denmark)diluted 5000-fold with T-TBS containing a 10% blocking solution(Blockace (trademark), DAINIPPON PHARMACEUTICAL CO., LTD. Japan) wasadded per well. The solutions were incubated at room temperature for 30minutes, so as to bind the streptavidin with the biotin-labeledantibodies. Each well was washed 4 times with T-TBS, and then 50 μl oftetramethylbenzidine (DAKO, Denmark), which is a peroxidase chromogenicsubstrate, was added per well, followed by incubation at roomtemperature. 30 minutes later, 50 μl of 0.5 M sulfuric acid solution wasadded per well, so as to stop reaction. Measurement was conducted usinga system for measuring absorbance for a 96-well plate, and values(A450-A570) were obtained by subtracting absorbance at 570 nm fromabsorbance at 450 nm. Concentrations were calculated by converting thevalues into values denoting concentrations of the purified products ofrecombinant human FGF-23 protein based on the calibration curve.

As a result, as shown in FIG. 22B, a significant increase was observedin the blood FGF-23 concentrations of the group to which feed mixed withadenine had been given. As shown in Example 24, administration of FGF-23to normal mice can cause a rapid decrease in blood 1,25D concentration,suggesting the presence of some relationship between a decrease in blood1,25D and an increase in blood FGF-23 concentration when renal functionsdecrease. To verify the possibility, blood 1,25D concentrations andblood FGF-23 concentrations after feeding of feed mixed with adeninewere plotted for individual rats. Thus, it was revealed that they show asignificant negative linear correlation (FIG. 22C). These resultssuggested the possibility that controlling blood FGF-23 concentrationscan correct decreases in blood 1,25D. Hence, an experiment ofadministration of antibodies neutralizing anti-FGF-23 to this model wasconducted in the manner described below.

Twelve 7-week-old male Wister rats were fed ad lititum for 7 days withmixed feed prepared by compounding adenine (6-aminopurine, Sigma,U.S.A.) to a final concentration of 0.75% with CE-2 commercial powderfeed for rodents (CLEA Japan, Inc., Japan), thereby producing a modelhaving decreased renal functions. To 12 mice of a control group, onlyCE-2 was fed. To make the degree of the disorder of renal functionsequivalent using a blood creatine level on day 7 after the production ofthe model, the rats of each group fed with feed mixed with adenine andthe rats of the control group were each divided into 2 groups (6rats/group), and then single administration of the 3C1E antibody wasperformed at 1 mg/kg or that of PBS (vehicle) was performed at 1 ml/kg,via the caudal vein. 12 hours later, partial blood collection wasperformed via the caudal artery, and then the blood was subjected tocentrifugation, thereby obtaining serum. 1,25D concentration in theobtained serum was measured using a 1,25(OH)2D RIA kit TFB (TFB,U.S.A.), and creatinine concentration in the obtained serum was measuredusing CRE-EN Kainos ((trademark), KAINOS LABORATORIES, INC., Japan).

As a result, as shown in FIG. 22D, in all of the control groups and thegroups fed with feed mixed with adenine, significant increases wereobserved in 1,25D concentrations compared with each group given with avehicle. The above results suggested that FGF-23 is involved inhypovitaminosis D (disease with low levels of 1,25D in blood)accompanying decreased renal functions, and at this time the suppressionof the action of FGF-23 can improve hypovitaminosis D.

EXAMPLE 30 Detection of Blood FGF-23 Protein in a X-LinkedHypophosphatemic Rickets (XLH) Patient

XLH is a form of hypophosphatemic rickets showing an X-linked inheritedcharacter, and the morbidity thereof shares many points in common withthat of tumor-induced osteomalacia. In recent years, phex belonging tothe metalloendopeptidase family has been identified as the causativegene of this disease, and a putative substrate thereof has beensuggested to be a factor inducing morbidity. In the meantime, as shownin Example 23, it was revealed that blood FGF-23 concentrations areextremely high in Hyp mice, which are hereditary hypophosphatemia modelmice having a gene mutation in phex that is the same as that in XLH.These phenomena suggested the possibility that hypophosphatemia in XLH,similarly to that in tumor-induced osteomalacia, is caused by elevatedblood FGF-23 concentrations. Hence, first, for the purpose of examiningthe involvement of FGF-23 in this disease, FGF-23 concentrations in theserum of normal subjects and XLH patients were measured. In addition,serum subjected to measurement had been provided after having obtainedthe sufficient prior approval of normal subjects and XLH patientsconfirmed to have gene mutations in phex. The age, gender, and mutationposition of the phex gene of the patients are described in FIG. 23.

In ELISA assay, the 2C3B antibody was used as an immobilized antibody,and the biotin-labeled 3C1E antibody was used as an antibody fordetection. First, the purified 2C3B antibody was diluted to 10 μg/mlwith a 50 mM sodium hydrogen carbonate solution. 50 μl of the resultantsolution was added per well of a 96-well plate for ELISA (Maxisorp(trademark), Nunc, U.S.A.), and then incubated at 4° C. for 12 hours forimmobilization. Subsequently, the reaction solutions were removed, 250μl of a blocking solution (SuperBlock (trademark), PIERCE, U.S.A.) wasadded per well, and then the resultant was incubated at room temperaturefor 30 minutes, thereby performing blocking. After the solutions wereremoved, the wells were washed 2 times with TBS (T-PBS) containing 0.1%Tween 20. The serum collected from XLH patients were prepared, anddiluted 2-fold with T-TBS containing a mouse IgG1 antibody not bindingto FGF-23 at a concentration of 100 μg/ml. In addition, standardproducts were diluted using the serum of normal subjects (from whichendogenous FGF-23 had been removed by subjecting the serum to theanti-2C3B-antibody-immobilized resins prepared in Example 14) diluted2-fold with T-TBS containing the mouse IgG1 antibody not binding toFGF-23 at a concentration of 100 μg/ml. 50 μl of each sample was addedper well of a microtiter plate, and then the samples were incubated atroom temperature for 1 hour, thereby causing the FGF-23 protein in theanalytes to react with the immobilized antibodies. After antibodyreaction, the wells were washed 4 times with T-TBS, and then thebiotin-labeled 3C1E antibody diluted to 1.5 μg/ml with T-TBS containinga 10% blocking solution (Blockace (trademark), DAINIPPON PHARMACEUTICALCO., LTD. Japan) was added. The solutions were incubated at roomtemperature for 30 minutes, thereby performing secondary antibodyreaction. After each well was washed 4 times with T-TBS, 50 μl ofHRP-labeled streptavidin (DAKO, Denmark) diluted 5000-fold with T-TBScontaining a 10% blocking solution (Blockace (trademark), DAINIPPONPHARMACEUTICAL CO., LTD. Japan) were added per well. The solutions wereincubated at room temperature for 30 minutes, so as to bind thestreptavidin with the biotin-labeled antibodies. Each well was washed 4times with T-TBS, and then 50 μl of tetramethylbenzidine (DAKO,Denmark), which is a peroxidase chromogenic substrate, was added perwell, followed by incubation at room temperature. 30 minutes later, 50μl of 0.5 M sulfuric acid solution was added per well, so as to stopreaction. Measurement was conducted using a system for measuringabsorbance for a 96-well plate, and values (A450-A595) were obtained bysubtracting absorbance at 595 nm from absorbance at 450 nm.

As a result, blood FGF-23 concentrations of 104 normal adults (30 menand 74 women) were aggregated in a range from 8.2 to 54.3 ng/l, and theaverage value and the standard error thereof were 28.9±1.1 ng/l. In themeantime, as shown in FIG. 23, all of the serum FGF-23 concentrations inXLH patients were higher than the average values for normal subjects,and even the average concentration in XLH patients was alsosignificantly higher than that of normal subjects (p<0.0001, calculatedby Student-t). These results strongly suggested the possibility thatFGF-23 at high concentrations in the serum of XLH patients acts as afactor inducing the disease.

EXAMPLE 31 Action Augmenting Neutralization Activity when 2 DifferentTypes of Neutralization Antibody are Mixed

Changes in physiological action when neutralization antibodiesrecognizing different sites were mixed were examined using normal mice.

8-week-old male C57BL/6 mice were randomly divided into 4 groups, eachconsisting of 6 mice. Single administration of PBS as a vehicle wasperformed per mouse via the caudal vein for group 1, that of 100 μg ofthe 2C3B antibody was performed per mouse via the caudal vein for group2, that of 100 μg of the 3C1E antibody was performed per mouse via thecaudal vein for group 3, and that of the admixture of antibodies (50 μgof the 2C3B antibody and 50 μg of the 3C1E antibody) was performed permouse via the caudal vein for group 4. On days 1 and 2 afteradministration, blood was collected from the eye sockets under etheranesthesia, and then serum was separated using a microtainer (BectonDickinson, U.S.A.). Phosphate concentrations in the thus obtained serumwere measured using P (phosphorus)-Test Wako (Wako Pure ChemicalIndustries, Ltd., Japan) according to the attached document. During theperiod from administration to blood collection, each group of mice waskept in a plastic cage, and fed ad libitum with CE-2 (CLEA Japan, Inc.,Japan) along with tap water.

The obtained results are shown in FIG. 24. 100 μg of the 2C3B antibodyor the 3C1E antibody alone caused a significant increase in serumphosphorus concentrations on day 1 after administration. On the otherhand, in the group to which the mixture of the antibodies had beenadministered, although the dose of each antibody had been reduced byhalf, action to significantly increase serum phosphorus concentrationswas shown, and significantly high serum phosphorus concentrations wereshown compared with any group to which a single type of antibody hadbeen administered. Furthermore, on day 2 after administration, whereasin the group to which the 2C3B antibody or the 3C1E antibody had beenadministered, action increasing serum phosphorus concentrationsdisappeared, serum phosphorus concentrations in the group to which themixture of the antibodies had been administered were still significantlyincreased. The above results showed that the mixture of 2 types ofantibodies recognizing different sites can further augmentneutralization activity and maintain the action time longer than incases where 1 type of antibody alone was administered.

EXAMPLE 32 Experiment of Administration of FGF-23 NeutralizationAntibodies to Hereditary Hypophosphatemia Model Mice

As shown in Examples 23 and 30, increases in blood FGF-23 concentrationswere shown in XLH and Hyp mice, suggesting the possibility that FGF-23is a responsible factor of hypophosphatemia, abnormal vitamin Dmetabolism, or disorders of bone calcification, which are morbidities ofthe diseases. Hence, anti-FGF-23 neutralization monoclonal antibodieswere administered to Hyp mice, and then whether or not the abovemorbidities could be improved was examined.

(1) Experiment of Repeated Administration of anti-FGF-23 NeutralizationAntibodies

Using 4- to 7-week-old male C57BL/6-Hyp mice and wild-type male C57BL/6mice in same littemates, effects by the repeated administration ofneutralization antibodies on blood phosphate concentrations and blood1,25D concentrations in Hyp mice were examined. Four groups establishedin this experiment were composed of a group of wild-type mice to whichvehicles were administered, a group of wild-type mice to whichantibodies were administered, a group of Hyp mice to which vehicles wereadministered, and a group of Hyp mice to which antibodies wereadministered. Each group consisted of 4 mice. In the case of the groupsto which antibodies were administered, specifically, the 3C1E antibodywas admixed with the 2C3B antibody, so that each of their finalconcentrations would be 1.7 mg/ml, and then the resultant wasadministered at a dose of 17 mg/kg subcutaneously to the dorsal regionof each mouse. 10 mg/kg PBS in a volume equivalent to that of antibodiesadministered to the groups to which the antibodies were administered wasadministered per mouse. On days 2, 4, and 6 after the initialadministration, similar administration was performed. On days 1 and 7after the initial administration, the ends of the tail portions wereincised, and then partial blood collection was performed under etheranesthesia. For the measurement of blood phosphate and 1,25Dconcentrations, as well as blood total alkaline phosphatase activity, P(phosphorus)-Test Wako (trademark, Wako Pure Chemical Industries, Ltd.,Japan), Calcium-Test Wako (trademark, Wako Pure Chemical Industries,Ltd., Japan), a 1,25(OH)2D RIA Kit TFB (TFB, U.S.A.), and AlkalinePhospha B-Test Wako (trademark, Wako Pure Chemical Industries, Ltd.,Japan) were used, respectively.

The results are shown in FIG. 25A. In the group of wild-type mice towhich the antibodies had been administered, significant increases wereobserved in serum phosphate concentrations during day 1 to day 7 afteradministration. In the group of Hyp mice to which the antibodies hadbeen administered, significant increases were also observed in bloodphosphate concentrations compared with those in the groups to whichvehicles had been administered on day 1 after administration. Theseincreases became more significant on day 7 after administration, showingconcentrations to the same degree as those of the group of wild-typemice to which the antibodies had been administered. Moreover, by theadministration of the neutralization antibodies, significant increaseswere observed in blood 1,25D concentrations on day 1 afteradministration in both wild-type mice and Hyp mice. In the meantime,abnormal increases in blood total alkaline phosphatase activity havebeen reported in Hyp mice or in patients with hyphophosphatemic ricketssuch as XLH. It was revealed that administration of the neutralizationantibodies to Hyp mice attenuated, from 24 hours after theadministration, this blood total alkaline phosphatase activity atabnormally higher levels than those in normal mice, and further causedthe activity to significantly decrease to the same degree as that inwild-type mice on day 7 after initial administration.

Using 4- to 7-week-old male C57BL/6J-Hyp mice (Hyp mice) and wild-typemale C57BL/6J mice (wild-type littermate mice) and of the normal controlgroup, effects of repeated administration of anti-FGF-23 neutralizationantibodies on the bone tissues of Hyp mice were examined. The fourgroups established in this experiment were: a group of wild-type mice towhich vehicles were administered, a group of wild-type mice to whichantibodies were administered, a group of Hyp mice to which a vehicle wasadministered, and a group of Hyp mice to which antibodies wereadministered. Each group consisted of 4 mice. In the case of the groupsto which antibodies were administered, specifically, the 3C1E antibodywas admixed with the 2C3B antibody, so that each of their finalconcentrations would be 1.7 mg/ml, and then the resultant wasadministered at a dose of 17 mg/kg subcutaneously to the dorsal regionof each mouse. In the case of the group of mice to which vehicles wereadministered, specifically, 10 ml/kg PBS was administered subcutaneouslyto the dorsal region of each mouse. When the day of the initialadministration is denoted day 0, on days 2, 4, 6, 8, 21, and 40, similaradministration was performed. On day 49 after the initialadministration, blood was collected from the heart under etheranesthesia, and right femur samples, and right tibia samples, costasamples were collected. X-ray magnified images of the excised femur,tibia, and costa samples were taken by X-ray irradiation using a μFFX-1000 Microfocus X-ray magnified imaging system (FUJI PHOTO FILM CO.,LTD., Japan) under outgoing conditions of 100 μA and 25 kV for 5seconds, and then obtained after digital imaging using a BAS imaginganalyzer (FUJI PHOTO FILM CO., LTD., Japan).

The thus obtained X-ray magnified images of extracted femora, tibiae,and costae are shown in FIG. 27. In Hyp mice or in the case ofhypophosphatemic rickets such as XLH, increased chondrocytes observed inassociation with calcification disorders and enlargement of themetaphysis of long bones or osseocartilaginous junction in costae havebeen reported. As a result of X-ray imaging analysis, alleviation wasrecognized in the group of Hyp mice to which the antibodies had beenadministered for enlargement of the metaphysis, decreased bone density,and thinning of cortical bones of the femora and tibia that wererecognized in the group of mice to which the vehicles were administered.Furthermore, alleviation was also recognized in the group of Hyp mice towhich the antibodies had been administered for failure of extension inthe longitudinal direction of femora and tibia observed in the group ofHyp mice to which the vehicle had been administered. Moreover,enlargement of the junctions of costae and of costal cartilages observedin the group of Hyp mice to which the vehicle had been administereddisappeared in the group of Hyp mice to which the antibodies had beenadministered. It was revealed that repeated administration ofanti-FGF-23 neutralization antibodies to Hyp mice improves disorders ofbone extension and disorders of bone calcification.

Furthermore, for the purpose of examining in detail the action of theanti-FGF-23 antibodies on bone tissues, bone tissue samples of animalssubjected to this experiment were prepared. After sacrifice, the lefttibiae and the left femora were extracted, fixed with 70% ethanol,subjected to Villanueva Bone staining (Villanueva Bone Stain Powder,MARUTO INSTRUMENT CO., LTD. Japan), dehydrated with ethanol solutionshaving sequentially elevated concentrations, and then embedded inTechnovit 7100 (Kulzer, Germany). The resultants were sectioned to havean approximate thickness of 5 μm, and then the sections of the proximalregion of the tibia and the distal region of the femora were observedunder a microscope. The images of the bone tissue samples are shown inFIG. 28. Whereas growth plates where chondrocytes are aligned in anorderly manner were observed in wild-type mice (FIGS. 28A and D), in thebone tissues of Hyp mice, regions with a disturbed column structure,characterized by enlarged regions of the growth plates and irregularsequences of chondrocytes (FIGS. 28B and E) were observed. In contrast,in Hyp mice to which the neutralization antibodies against FGF-23 hadbeen repeatedly administered, suppressed enlargement of the width of thegrowth plate and disappearance of the irregular sequences of chondrocytswere observed (FIGS. 28C and F).

(2) Experiment of Single Administration of Anti-FGF-23 NeutralizationAntibodies

Using 12- to 20-week-old male C57BL/6-Hyp mice and wild-type maleC57BL/6 littermate mice, effects of single administration ofneutralization antibodies on blood phosphate concentrations and blood1,25D concentrations in Hyp mice were examined. The four groupsestablished in this experiment were: a group (n=4) of wild-type mice towhich a vehicle was administered, a group (n=5) of wild-type mice towhich antibodies were administered, a group (n=3) of Hyp mice to which avehicle were administered, and a group (n=3) of Hyp mice to whichantibodies were administered. In the case of the groups to whichantibodies were administered, specifically, the 3C1E antibody wasadmixed with the 2C3B antibody, so that each of their finalconcentrations would be 1.7 mg/ml, and then the resultant wasadministered once at a dose of 17 mg/kg subcutaneously to the dorsalregion of each mouse. In the case of the group of mice to which avehicle was administered, specifically, PBS was administered at a doseof 10 ml/kg. On day 4 after administration, blood was collected from thehearts and the kidneys were extracted under ether anesthesia.

The thus obtained results of measuring serum phosphate and 1,25Dconcentrations are shown in FIG. 25B. By the single administration ofthe antibodies to wild-type mice, both serum phosphate and 1,25D showedsignificant increases on day 4. This effect of increasing theconcentrations was also observed in Hyp mice. By the administration ofthe antibodies, the serum phosphate concentration was corrected from thelowered level to the normal level and the 1,25D concentration showed asignificant increase.

(3) Effect of Controlling the Expression of NaPi2a and 1αOHase byAnti-FGF-23 Neutralization Antibodies

A serum phosphate concentration is determined by the reabsorption rateby sodium-dependent phosphate cotransporters (NaPi2a) that are mainlypresent in the renal proximal tubule. Furthermore, it has already beenreported that the reabsorption rate is controlled depending on theexpression level of NaPi2a itself or the localization ratio of NaPi2aprotein. Hence, whether or not the effect of anti-FGF-23 neutralizationantibodies to increase serum phosphate concentrations is mediated byNaPi2a was examined.

Renal proximal tubule brush border membrane vesicles (BBMV) where theNaPi2a was localized were prepared using a kidney subjected tofreeze-thawing, according to a calcium precipitation method aspreviously reported by Katai et al. in J. Biol. Chem. 274, pp.28845-28848, 1999. 20 μg of each obtained BBMV was subjected to Westernblotting according to the method described in Example 16. The NaPi2aprotein was detected using anti-mouse NaPi2a rabbit polyclonalantibodies purified from anti-serum that had been obtained by immunizingrabbits similarly to the case of Example 5 with the C-terminal partialpeptide (SEQ ID NO: 32) of a mouse NaPi2a protein. The results are shownin FIG. 25C. It has already been reported that NaPi2a protein levels aresignificantly lowered in the BBMV of Hyp mice. However, by theadministration of anti-FGF-23 neutralization antibodies, the expressionof the NaPi2a was significantly increased.

Also in the wild-type mice, further increase was observed in the NaPi2aprotein concentrations. Moreover, the phosphate transport activity bythe NaPi2a protein was measured using the above BBMV. Using 0.1 mg ofeach BBMV prepared from each individual, sodium-dependentphosphate-uptake activity for 60 seconds was measured by a rapidfiltration method. Reagent and reaction conditions were employedaccording to the method as reported by Katai et al. in J. Biol. Chem.274, pp. 28845-28848, 1999.

As a result, as shown in FIG. 25C, increases in phosphate transportactivity resulting from the administration of the antibodies wereobserved in both wild-type mice and Hyp mice. These results suggestedthat the action of increasing serum phosphate concentrations inwild-type mice or Hyp mice by the anti-FGF-23 neutralization antibodiesis caused by increase of the levels of NaPi2a protein existing in therenal proximal tubule. mNpt2C: LALPAHHNATRLC (SEQ ID NO: 32)

Next, to elucidate the mechanism of action to increase serum 1,25Dconcentrations, the expression pattern of kidney 25 hydroxyvitamin D-1αhydroxylase (1αOHase) was analyzed. Total RNA was extracted using anISOGEN reagent (NIPPON GENE CO., LTD., Japan) from a frozen kidney. 20μg of each obtained RNA was subjected to Northern blotting according tothe standard method. A PerfectHyb reagent (Toyobo Co., Ltd., Japan) wasused for hybridization, and reaction and washing of blots were conductedaccording to the attached documents. A probe used herein was prepared byamplifying a mouse 1αOHase partial cDNA by the PCR method using cDNAlibrary prepared from a mouse kidney as a template, and oligonucleotidesrepresented by SEQ ID NOS: 33 and 34 as primers, or amplifying as aninternal standard a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)partial cDNA by the PCR method using oligonucleotides represented by SEQID NOS: 35 and 36 as primers, and then labeling the cDNA with ³²P usinga Megaprime DNA labeling system (Amersham bioscience, U.S.A.). Signaldetection was performed using a BAS imaging analyzer (FUJI PHOTO FILMCO., LTD., Japan). The results are shown in FIG. 25D. By theadministration of the neutralization antibodies, significantly enhancedexpression of 1αOHase was confirmed in wild-type mice and Hyp mice.These results suggested that the action of the anti-FGF-23neutralization antibodies to increase serum 1,25D concentrations inwild-type mice or Hyp mice was due to enhanced gene expression levels of1αOHase existing in the kidney.

-   -   m1alphaFW: cagacagagacatccgtgtag (SEQ ID NO: 33)    -   m1alphaRV: ccacatggtccaggttcagtc (SEQ ID NO: 34)    -   gapdhFW: accacagtccatgccatcac (SEQ ID NO: 35)    -   gapdhRV: tccaccaccctgttgctgta (SEQ ID NO: 36)

The above results suggested that FGF-23 is strongly involved indecreases in blood phosphate concentrations and blood 1,25Dconcentrations in Hyp mice, that is, in the case of human XLH, andsuggested the possibility that inhibition of the FGF-23 action by theadministration of the anti-FGF-23 antibodies can improve the morbiditythereof. Moreover, abnormally high levels of blood total alkalinephosphatase activity in Hyp mice along with disorders of bone extensionand disorders of bone calcification were also normalized by theadministration of the anti-FGF-23 antibodies. Hence, remission ofosteomalacia accompanied by normalization of bone remodeling isexpected.

EXAMPLE 33 Action of Anti-FGF-23 Antibody (3C1E) on Bone Remolding

As described in Example 32(1), the action of the anti-FGF-23 antibodieson bone remodeling was suggested. To further examine this suggestion,changes in serum osteocalcin concentrations after administration of theanti-FGF-23 antibodies were analyzed with time. The experiment wasconducted as follows. Single administration of the 3C1E antibody at 3mg/kg, or that of vehicles (PBS) at 1.1 ml/kg was performed via thecaudal veins of 7-week-old male S.D. rats. At 4, 12, 24, 48, and 72hours after administration, blood was partially collected from thecaudal arteries, and then serum was prepared. Serum osteocalcinconcentrations were measured using an osteocalcin ELISA kit (Amershambioscience, Japan). The results are shown in FIG. 26. At 12, 24, and 48hours after administration of the antibodies, significant increases wereobserved in serum osteocalcin concentrations. These results suggestedthe possibility that the administration of the anti-FGF-23 antibodiescan have a direct or indirect effect on bone remodeling.

EXAMPLE 34 Action of Anti-FGF-23 Neutralization Antibodies (Mixture of2C3B and 3C1E Antibody) on Osteoporosis Model Mice

For the purpose of further examining the action of the anti-FGF-23neutralization antibodies on bone metabolism as described in Example 33,the action of the anti-FGF-23 antibodies on post-ovariectomyreduced-bone-mass model mice that were thought to reflect reduced bonemass observed in the case of postmenopausal osteoporosis was examined.Ovaries were extracted from 8-week-old female ddy mice, and the micewere divided into 2 groups: a group to which a vehicle was administered,and a group to which the anti-FGF-23 antibodies were administered. Inaddition, as an operation control group, a sham operation groupsubjected to sham operation was established. For the group (n=10) towhich the antibodies were administered, administration was begun on day3 after operation, and anti-FGF-23 antibodies prepared by mixing anequivalent volume of the 2C3B antibody and the 3C1E antibody (3 mg/kg;1.5 mg/kg each of the 2C3B antibody and the 3C1E antibody) wereadministered subcutaneously to the dorsal region of the mice 3 times aweek for 4 weeks. Furthermore, to the group (n=9) of mice to which avehicle was administered and the group (n=10) of mice which weresubjected to sham operation, PBS was administered as a vehicle in thesame quantity (10 ml/kg) as that of the solution of the antibodies. Onweek 2 after ovariectomy, blood was collected from the eye sockets underether anesthesia, and on week 4 after ovariectomy, blood was collectedfrom the hearts under ether anesthesia. Serum was prepared from thecollected blood by centrifugation at 8000 rpm×10 minutes, and thensubjected to the measurement of serum osteocalcin (IRMA kit, Immutopics,Inc.). After sacrifice by collecting blood from the hearts on week 4after ovariectomy, the femora were extracted, and then bone mineralcontent and bone mineral densities (Bone Densitometer, Model DCS-600,ALOKA CO., LTD.) were measured.

As shown in FIGS. 29A and B, in both cases on weeks 2 and 4 afterovariectomy, significantly increased serum osteocalcin levels were wasobserved in the group to which the anti-FGF-23 antibodies (the mixtureof 2C3B and 3C1E) had been repeatedly administered, compared with thosein the group to which the vehicle had been administered. Osteocalcin isa protein that is specifically produced by osteoblasts. Since elevatedlevels of serum osteocalcin in vivo are thought to be an indicator ofbone formation, a possibility was shown that the administration of theanti-FGF-23 antibodies promotes bone formation.

Moreover, as shown in FIGS. 30A and B, the group which had beensubjected to overiectomy and to which the vehicle had been administeredshowed significantly reduced bone quantity compared with the group whichhad been subjected to sham operation and to which the vehicle had beenadministered. In contrast to the significantly decreased bone mineralcontent and bone mineral density by ovariectomy, the group which hadbeen subjected to overiectomy and to which the anti-FGF-23 antibodieshad been administered showed significantly high levels of bone mineralcontent and bone mineral density. Based on these results, it wasconsidered that the administration of anti-FGF-23 antibodies suppressedthe reduction in bone quantity resulting from overiectomy.

EXAMPLE 35 Experiment of Administration of Anti-FGF-23 NeutralizationAntibodies (Mixture of 2C3B Antibody and 3C1E Antibody) to HereditaryHypophosphatemic Rickets Model Mice

As shown in Example 32, the anti-FGF-23 neutralization antibodies wereshown to alleviate disorders of bone extension and disorders of bonecalcification in Hyp mice. Hence, the action of the above anti-FGF-23neutralization antibodies on disorders of bone extension and disordersof bone calcification in Hyp mice was further examined in detail using4-week-old male C57BL/6-Hyp mice and wild-type male C57BL/6 littermatemice that were younger in week age than mice used in Example 32, andthus were growing significantly. Four groups established in thisexperiment were: a group of wild-type mice to which a vehicle (PBS) wasadministered, a group of Hyp mice to which a vehicle (PBS) wasadministered, a group of Hyp mice to which antibodies were administeredat a low dose, and a group of Hyp mice to which antibodies wereadministered at a high dose. Each group consisted of 6 to 7 mice. Asneutralization antibodies, a solution containing a mixture of the 2C3Bantibody and the 3C1E antibody at the same concentration to each otherwas used. The solution was prepared so that the total quantity of the 2types of antibodies was 4 mg/kg for the low-dose group and 16 mg/kg forthe high-dose group. The neutralization antibodies or the vehicle wasrepeatedly administered subcutaneously at a volume of 10 mL/kg to thedorsal region of each mouse on days 7, 14, 21, and 28 when the day ofthe initial administration was denoted day 0. Body weight, the length ofthe tail, and thel length of the tibia were measured on the day same asthe day on which the antibodies were administered. Furthermore, on day31 after the initial administration, mice were sacrificed by collectingblood from the hearts under ether anesthesia, and then the femora andthe tibiae on the left and the right were extracted.

(1) Action of Anti-FGF-23 Neutralization Antibodies to Extend the Lengthof the Tail and the Length of Tibia and to Increase Body Weight

The bones of Hyp mice present rachitic bone phenotypes accompanied bycharacteristic impaired bone development such as disorders of extensionof long bones in the longitudinal direction. As an individual mouse, anHyp mouse is obviously shorter in body length and of lower in bodyweight than wild-type mice. To examine the effect of the administrationof the anti-FGF-23 neutralization antibodies on such impaired bonedevelopment in Hyp mice, measurement of the length of the tail portionand the length of the tibia and changes in body weight in Hyp mice usedin the experiment were examined. The results are shown in FIGS. 31A, B,and C. Also in this experiment, in contrast to normal mice, Hyp micepresented significantly low degrees of extension of the tail portionsand of tibiae, and low body weights. Furthermore, compared with thegroup of Hyp mice to which the vehicle had been administered, theneutralization antibodies administered to Hyp mice exerted action toextend the length of the tail portions and the length of the tibiae andaction to increase body weights in a dose-dependent manner.

(2) Bone Calcification Action by Anti-FGF-23 Neutralization Antibodies

The bone tissue of Hyp mice is known to be rich in terms of theproportions of uncalcified and low-calcified bone, but in poor inmineral content compared with the case of a normal mouse. Hence, it wasexamined whether or not action of the anti-FGF-23 neutralizationantibodies to increase the bone mineral content in the Hyp mouse femoracould be obtained. In this experiment, the extracted left femora weredried with a dryer at 100° C. for 12 hours, and then weighed, therebyobtaining the dried bone weight. Next, the dried bone was incinerated byheating in a muffle furnace at 600° C. for 12 hours. The resultants wereweighed so as to obtain bone ash weights. The results are shown in FIG.32. The ratio of bone ash weight to dried bone weight was very low inHyp mice, compared with the case of wild-type mice. The ratio of boneash weight to dried bone weight in the group of Hyp mice to which theantibodies had been administered significantly increased in a mannerdepending on the dose of the neutralization antibodies repeatedlyadministered. In particular, the group to which the antibodies had beenadministered at a high dose showed improvement to the extent of showingvalues at almost the same levels as those in the group of wild-typemice.

These results reveled that inhibiting the action of FGF-23 by theadministration of anti-FGF-23 neutralization antibodies can improvedisorders of bone extension and disorders of bone calcificationpresented by Hyp mice.

EXAMPLE 36) Quantitative measurement of human FGF-23 protein by sandwichELISA method using 2C5L antibody

The 2C5L antibody produced by the 2C5L cloned hybridoma obtained inExample 3 was purified according to Example 4. Analysis of therecognition site thereof by the method described in Example 10 revealedthat the 2C5L antibody has a recognition site within the N-terminal sidefragment polypeptide corresponding to a region between the 25^(th) andthe 179^(th) amino acid residues of SEQ ID NO: 1 in the same manner asthe 2C3B antibody, and forms an immunocomplex with the N-terminal sidefragment polypeptide and a full-length FGF-23 protein. Moreover, in amanner described in Example 12, establishment of a sandwich ELISA methodusing a combination of the 2C5L antibody and the 2C3B antibody wasattempted. Upon binding of each antibody with the FGF-23 protein, bothantibodies were shown not to competitively inhibit each other. Accordingto this result, the 2C5L antibody has a recognition site within theN-terminal side fragment polypeptide corresponding to a region betweenthe 25^(th) to 179^(th) amino acid residues, but the recognition sitethereof is different from that of the 2C3B antibody having a recognitionsite also within the N-terminal side fragment polypeptide. This revealedthat an ELISA method using a combination of the two types of antibodiesis possible. Next, by the method described in Example 13, the ability todetect FGF-23 protein was examined when biotin-labeled 3C1E antibody wasused as antibodies for detection, and the 2C3B or 2C5L antibodies wereused as immobilized antibodies. The results are shown in FIG. 33. It wasconfirmed that the 2C5L antibody, in concentrations ranging from 10pg/ml to 10 ng/ml, recognizes purified products of recombinant humanFGF-23 in a concentration-dependent manner.

EXAMPLE 37 Cross Reactivity Between 2C5L Antibody and Mouse FGF-23

In the sandwich ELISA system using a combination of the 2C3B antibodyand the 3C1E antibody as shown in Examples 22 and 23, mouse FGF-23 couldalso been recognized similarly to human FGF-23. Hence, whether or notthe 2C5L antibody could recognize mouse FGF-23 was examined. Similarlyto Example 24, mouse serum with high levels of blood FGF-23 was obtainedand then measured with a combination of the 2C3B and biotin-labeled 3C1Eantibody, thereby obtaining a sample showing approximately 600 pg/ml.For this sample, a sandwich ELISA system similar to that in Example 36was examined using a combination of the 2C5L antibody and thebiotin-labeled 3C1E antibody. However, no reactivity was observed. Thisresult showed that the 2C5L antibody has strong binding ability to humanFGF-23, but has very weak or no binding ability to mouse FGF-23.

EXAMPLE 38 Measurement of Ability of Neutralization Activity of 2C5LAntibody for Human FGF-23

The results in Example 37 suggested that since the binding ability ofthe 2C5L antibody to mouse FGF-23 is very low compared with that of the2C3B antibody or the 3C1E antibody, the ability of neutralizationactivity of the 2C5L antibody for mouse endogenous FGF-23 would also notbe significant. However, according to the result in Example 36, sincethe 2C5L antibody has strong binding ability to human FGF-23, the 2C5Lantibody may have the ability of neutralization activity for humanendogenous FGF-23. To verify the possibility, a system for measuringactivity to neutralize human FGF-23 using animals was constructed by thefollowing procedures, and then the activity of the 2C5L antibody toneutralize human endogenous FGF-23 was examined using this system.

An osmotic mini pump (alzet micro-osmotic pump model 1007D, DURECT,Canada) that had been adjusted to gradually release human FGF-23recombinants at a dose of 1.2 μg/day was transplanted subcutaneouslyinto the dorsal region of a 7-week-old male C57BL/6 mouse, therebyconstructing a model to which the human FGF-23 recombinant wascontinuously administered. An anti-FGF-23 neutralization antibodycontaining 2C5L or a vehicle was administered intraperitoneally on day 3after the transplantation of the pump, and then whether or not theaction of the continuously existing human FGF-23 recombinant to decreaseserum phosphate concentration could be suppressed was examined. The fourgroups established in this experiment were: a group (untreated/vehicle)of untreated mice to which a vehicle was administered, a group(FGF-23/vehicle) of thehuman-FGF-23-recombinant-continuously-administered model mice to which avehicle was administered, a group (FGF-23/2C5L) of thehuman-FGF-23-recombinant-continuously-administered model mice to whichthe 2C5L antibody was administered, and a group (FGF-23/2C3B) of thehuman-FGF-23-recombinant-continuously-administered model mice to whichthe 2C3B antibody was administered. PBS was used as vehicle and fordiluting the antibody. Anti-FGF-23 neutralization antibodies wereadministered at a dose of 20 mg/kg. The vehicle and the neutralizationantibody were administered at a volume of 0.1 mL per 10 g of bodyweight. Before antibody administration and 24 hours after antibodyadministration, blood was collected from the eye sockets under etheranesthesia, so as to obtain serum. Serum phosphate concentration wasmeasured using P-Test Wako (trademark, Wako Pure Chemical Industries,Ltd., Japan).

The results are as shown below. By the continuous administration of thehuman FGF-23 recombinants using the osmotic mini pump, on day 3 aftertransplantation of the pump, a significant decrease in blood phosphorusconcentration was recognized compared with the group of untreated miceto which the vehicle had been administered (FIG. 34, left). When the2C5L antibody was administered to thehuman-FGF-23-recombinant-administered group (FGF-23/2C5L group), 24hours later, the action to decrease serum phosphate concentration wassignificantly suppressed compared with the FGF-23/vehicle-administeredgroup or that before antibody administration. The concentration wasalleviated to the same degree as that of the serum phosphorusconcentration of the untreated/vehicle-administered group (FIG. 34,right). According to the above results and results in Example 37, itbecame clear that the 2C5L antibody exhibited neutralization activityagainst the action of decreasing blood phosphate depending on thecontinuously administered human FGF-23 recombinants. On the other hand,in the group to which the 2C3B antibody had been administered, the serumphosphorus concentration 24 hours later was significantly higher thanthat of untreated/vehicle-administered group. This result may be due tothe fact that the 2C3B antibody having strong binding ability with mouseFGF-23 exhibited neutralization activity not only with regard to theadministered human FGF-23 recombinant, but also with regard toendogenous mouse FGF-23.

Industrial Applicability

According to the present invention, antibodies against FGF-23 areprovided. The antibodies of the present invention are useful indiagnosing diseases or pathological conditions accompanying accumulationor decreases of the FGF-23 protein by appropriately detecting andmeasuring FGF-23 in vivo. Furthermore, the antibodies of the presentinvention have activity of neutralizing the action of FGF-23, so as tobe able to treat or alleviate diseases or pathological conditionsresulting from excessive action of FGF-23 by suppressing the action ofFGF-23.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety. It will be readilyapparent to persons skilled in the art that certain changes andmodifications may be made without departing from the technical idea orscope of the invention. The present invention intends to encompass suchchanges and modifications.

1. An antibody obtained by immunizing an animal with polypeptides whichcomprise an amino acid sequence represented by SEQ ID NO: 1, or an aminoacid sequence derived from the amino acid sequence represented by SEQ IDNO: 1 by deletion, substitution, or addition of 1 or several aminoacids, and has fibroblast growth factor-23 activity, which has activitycontrolling phosphate metabolism or vitamin D metabolism, and is shownby the following (a), (b), or (c): (a) an antibody which recognizes anamino acid sequence between the 180th and the 194^(th), or the 237^(th)and the 251^(st) amino acid residues represented by SEQ ID NO: 1; (b) anantibody which is produced by a hybridoma whose accession number is FERMBP-7838, FERM BP-7839, FERM BP-7840, or FERM BP-8268; or (c) an antibodywhich is competitive with the antibody produced by the hybridoma whoseaccession number is FERM BP-7838, FERM BP-7839, FERM BP-7840, or FERMBP-8268 upon binding with the polypeptide consisting of the amino acidsequence represented by SEQ ID NO:
 1. 2. The antibody of claim 1, whichis a monoclonal antibody.
 3. The antibody of claim 1 or 2, which isproduced by a hybridoma whose accession number is FERM BP-7838, FERMBP-7839, FERM BP-7840, or FERM BP-8268.
 4. A pharmaceutical composition,which comprises the antibody of any one of claims 1 to 3 as an activeingredient.
 5. The pharmaceutical composition of claim 4, which iseffective against at least one disease selected from tumor-inducedosteomalacia, ADHR, XLH, renal osteodystrophia, dialysis osteopathy,osteoporosis, hypophosphatemia, rickets, osteomalacia, dysfunction ofthe renal tubule, osteopenia, hypocalcemia, disorder of bone extension,disorder of bone calcification, hyperparathyroidism, ectopiccalcification, itching, osteosclerosis, Paget's disease, hypercalcemia,hypoparathyroidism, ostealgia, decreased muscle force, skeletaldeformation, failure to thrive, and hypovitaminosis D.
 6. Thepharmaceutical composition of claim 5, which is effective against atleast one disease selected from tumor-induced osteomalacia, XLH,hypophosphatemia, osteoporosis, disorder of bone extension, disorder ofbone calcification, and osteomalacia.
 7. An agent for promotingosteogenesis, comprising the antibody of any one of claims 1 to 3 as anactive ingredient.
 8. The pharmaceutical composition of any one ofclaims 4 to 6, which comprises at least 2 types of the antibodies ofclaim 1 recognizing different sites.
 9. The pharmaceutical compositionof claim 4, wherein the antibody recognizes an amino acid sequencebetween the 180^(th) and the 194^(th) amino acid residues represented bySEQ ID NO:
 1. 10. A method for detection of a fibroblast growthfactor-23, which comprises causing an antibody that recognizes a part ofan amino acid sequence between the 25^(th) and the 179^(th) amino acidresidues represented by SEQ ID NO: 1 and an antibody that recognizes apart of an amino acid sequence between the 180^(th) and the 251^(st)amino acid residues represented by SEQ ID NO: 1 to react with a testsample.
 11. The method for detection of claim 10, wherein the antibodythat recognizes a part of an amino acid sequence between the 180^(th)and the 251^(st) amino acid residues represented by SEQ ID NO: 1 is anantibody that recognizes an amino acid sequence between the 180^(th) andthe 196^(th) amino acid residues represented by SEQ ID NO:
 1. 12. Themethod for detection of claim 10, which uses a thrombin inhibitor. 13.The method for detection of claim 10 or 11, wherein the antibody isproduced by a hybridoma whose accession number is FERM BP-7838, FERMBP-7839, FERM BP-7840, or FERM BP-8268.
 14. A kit for detecting afibroblast growth factor-23, which contains an antibody that recognizesa part of the amino acid sequence between the 25^(th) and the 179^(th)amino acid residues represented by SEQ ID NO: 1 and an antibody thatrecognizes a part of the amino acid sequence between the 180^(th) andthe 251^(st) amino acid residues represented by SEQ ID NO:
 1. 15. Thekit of claim 14, wherein the antibody that recognizes a part of theamino acid sequence between the 180^(th) and the 251^(st) amino acidresidues represented by SEQ ID NO: 1 is an antibody that recognizes theamino acid sequence between the 180^(th) and the 196^(th) amino acidresidues represented by SEQ ID NO:
 1. 16. The kit of claim 14 or 15,wherein the antibody is produced by a hybridoma whose accession numberis FERM BP-7838, FERM BP-7839, FERM BP-7840, or FERM BP-8268.
 17. Ananti-fibroblast growth factor-23 antibody-binding material, to which atleast one antibody selected from the antibodies of claims 1 to 3 isbound.
 18. A medical appliance, which is provided with the bindingmaterial of claim
 17. 19. The medical appliance of claim 18, which isused for removing the fibroblast growth factor-23 in blood.